Potential Health Risks of Exposure to Graphene and Its Derivatives: A Review

Abstract

Graphene and its derivatives (GDs) have been applied in many fields, like photocatalysts, sensors, and biomedical delivery, due to its excellent physicochemical properties. However, the widespread use of GDs has significantly increased human exposure to these materials. Some health risks of exposure to GDs have been identified, including organ fibrosis, inflammation, DNA damage, etc. Given that graphene is a novel concern, we especially emphasized the various exposure pathways and potential health risks of exposure to GDs. People get exposed to GDs mainly through inhalation, ingestion, dermal contact, etc. GDs could transfer to the circular system of people and accumulate in blood, cells, and major organs. GDs exposure could induce organ and cell inflammatory responses and damage, such as disrupted kidney function, declined cell vitality, cytotoxicity, etc. These changes at the organ and cell levels might lead to adverse tangible influences on people, like decreased locomotor activity, the accelerated aging process, and even abnormal offspring development. We also summarized the characterization and detection methods of GDs. In addition, we compared the studies of exposure to dust and GDs in the aspects of health risks and study methods. This review could offer a comprehensive summary related to GDs and provide helpful references for further graphene-related studies.

1. Introduction

Graphene, with its exceptional properties, has emerged as a promising material for diverse applications across various industries, including food packaging, agriculture, electronics, machinery, optics, and clothing manufacturing [1,2]. These properties of graphene and its derivatives depend on factors such as their size, defects (cracks/folds/voids/wrinkles), thickness, number of layers, and functional groups. Identifying and characterizing the physical and structural properties of graphene are crucial for the application and research of graphene and its derivatives (GDs) [3,4].

Since the first observation of graphene under an electron microscope in 1962 [5], scholars have actively employed various methods to characterize GDs. The development of microscope technology has enabled the observation of graphene’s micromorphology, structure, and even the number of layers. The application of spectroscopic techniques allows for qualitative analysis of GDs. Various microscopes and spectroscopic technologies play significant roles in differentiating graphene materials, verifying the preparation of graphene-based materials, and monitoring the synthesis process of graphene.

The widespread application of graphene materials in multiple areas of life may result in their release into the environment and their entry into the human body and other organisms through contaminated water or food, raising concerns about their biological exposure risks and environmental impacts. For example, graphene is synthesized through various methods, including wet-chemical techniques and physical deposition techniques, which both generate waste that can potentially harm ecosystems if not properly managed [6]. Wet-chemical synthesis techniques, such as Hummers’ method, are commonly used to produce graphene oxide (GO) and reduced graphene oxide (rGO), meanwhile generating waste, including chemical byproducts (strong acids, alkalis, oxidizing agents, etc.), solid residues like salts, and graphene oxide particles. Physical deposition techniques, such as chemical vapor deposition (CVD) and physical vapor deposition (PVD), are widely used for producing high-quality graphene films, in which some gaseous byproducts and solid waste would be generated [6]. If waste is not treated before disposal, graphene particles, toxic chemicals, or byproducts can leach into water systems, affecting aquatic organisms and potentially entering the human food chain. Gaseous emissions from the physical deposition process, including CO₂ and methane, can contribute to air pollution and global warming. Nano-sized particles of graphene or byproducts may also be released into the atmosphere, posing inhalation risks for nearby populations. And improper disposal of solid wastes can result in soil contamination, leading to long-term environmental degradation and bioaccumulation of toxic substances in plants and animals [7].

Unlike the preparation and characterization of chemical materials, detecting graphene materials within cellular/biological tissues using analytical tools poses significant challenges due to interference from biological substances or low contrast between graphene and biological tissues [8]. In tracking the accumulation, distribution, migration, and transformation of graphene within organisms, isotopic labeling [9] emerges as a solution. It boasts strong tracing capabilities and accurate quantitation, providing information on the content, distribution, and behavior of graphene within organisms.

While current exposure levels in most workplaces may not be a significant concern due to the relatively low level of commercialization, as graphene and its derivatives become more widely used, the potential risks associated with exposure should not be underestimated. Graphene exposure can occur through multiple pathways, such as inhalation in occupational settings, oral ingestion via bioaccumulation in nanodrugs, skin contact, and intraperitoneal and intravenous administration in the biomedical field. These exposures have raised concerns about potential adverse health effects, including irritation-induced asthma, alterations in gene and protein expression, and disruptions in intestinal flora [8,10]. Studies using in vivo and in vitro methods have shown that GO and rGO can produce toxic effects on nerve cells and periodontal cells in a dose- and time-dependent manner, leading to apoptosis and other cellular responses [11].

Furthermore, graphene exposure has been linked to organ damage, like fibrosis, inflammation, granuloma formation, and cell damage. The fibrosis is a condition in which organ tissue becomes scarred, making it difficult for the lungs to function properly [12]. Inflammatory responses occur as the body recognizes inhaled or implanted graphene as a foreign substance, which leads to the activation of the immune system. Prolonged inflammation can worsen tissue damage and promote the development of diseases like asthma, chronic obstructive pulmonary disease (COPD), and other respiratory conditions [13]. Granulomas are clusters of immune cells that form around foreign substances that the body is unable to eliminate, which could impair organ function if they persist or grow in number [14]. The dysfunction, histopathological alternations, and fibrosis in the lung, liver, and kidney tissues were observed in mice intranasally administered with graphene quantum dots (0.1 or 1 mg/kg weight) [15]. A short-term decrease in locomotor activity and neuromuscular coordination of mice occurred after 5-day oral exposure to a high level of rGO (60 mg/kg body weight/d) nanosheets [10]. Longer-term exposure to FLG and GO at higher concentrations could result in reduced mitochondrial activity and more cytotoxic effects at the skin level [16]. As the use of graphene in consumer products continues to grow, the release of graphene into the environment through various pathways is inevitable, posing potential risks to terrestrial and aquatic organisms as well as human health. Therefore, it is necessary to figure out the potential health risks of exposure to GFMs, especially for human beings.

Both GDs and dust are inhalable particles in the atmosphere. Graphene and its derivatives vary in size from a few nanometers to hundreds of micrometers, exhibiting higher reactivity at the nanoscale due to an increased surface-to-volume ratio [17,18,19]. Its exposure comes primarily from nanomaterials used in electronics, batteries, composites, coatings, and medical applications. It is also produced during the manufacturing process of nanomaterials. While dust varies widely in size, typically between 0.1 µm and 100 µm [20]. Dust exposure is typically associated with industrial activities (mining, construction, manufacturing), agriculture, woodworking, transportation (vehicle emissions), and natural sources (sandstorms, volcanic activity). In addition, inhalation is the primary route of exposure to graphene and dust, though ingestion and dermal contact are also potential pathways [21,22,23]. Unlike GDs, dust exposure has been a long-standing public health issue. Thus, we compared the differences between the studies of dust and graphene, including health risks to people and research methods, which could provide more references for the further study of GDs.

In this review, we summarized the properties and applications of graphene. The characterization methods of graphene and methods for tracking graphene biological exposure were also addressed. We emphasized the various exposure pathways to GDs and their harmful influences on human beings. Additionally, we compared the studies of exposure to dust and GDs in their study methods and health risks to provide references for further graphene-related research.

2. Detection and Characterization of Graphene

2.1. Characterization of Graphene

The detection techniques for graphene and its derivatives are diverse, with characterization methods mainly divided into two categories: microscope observation and spectroscopic analysis. Microscope observation includes Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), and Scanning Tunneling Microscopy (STM). Among the spectroscopic techniques, Raman spectroscopy, X-ray Diffraction (XRD), and Ultraviolet-Visible spectroscopy (UV-Vis) are more frequently used. These characterization methods involve the study of graphene’s morphology, properties, defects, and layers.

Scanning electron microscopy (SEM) is one of the most common approaches to the characterization of graphene. SEM utilizes a high-energy electron beam to scan the surface of a sample point by point, acquiring morphology and structural information of the sample surface by detecting various signals generated from the interaction between the electrons and the sample, such as secondary electrons, backscattered electrons, and X-rays. It has a wide range of applications in detecting impurities and defects such as folds, voids, and wrinkles in graphene layers. At the same time, SEM plays a significant role in exploring the growth mysteries of graphene [24,25,26] and revealing the principles behind its corrosion resistance failure [27]. When measuring the number of graphene layers [28,29], the color darkens as the number of graphene layers increases in SEM imaging [30]. In SEM imaging, the color darkens as the number of graphene layers increases [30]. Many features in atomically thick graphene layers are difficult to image due to poor image contrast under normal imaging conditions, limiting the resolution in ultra-thin graphene layers. Adjusting the working distance and accelerating voltage during SEM imaging can enhance the image contrast of graphene [29,31].

TEM (Transmission Electron Microscope) also utilizes the interaction between an electron beam and the material being studied to form images. High-voltage electrons transmit through a thin sample, and the emitted photons form a TEM image on a phosphor viewing screen. In microscopic images, transparent sheets of single-layer graphene can be seen. In the case of multilayer graphene, multiple dark lines can be observed [32]. Despite having higher resolution for observing the lattice structure and atomic arrangement of graphene [33], it is important to note that TEM requires thinner sample preparation to allow high-voltage electrons to pass through. This is crucial for TEM imaging, as thicker samples may obscure the details of the graphene structure, making it difficult to obtain clear and accurate images. The following diagram illustrates the characterization of synthesized graphene-mesoporous silica-gold NP hybrids (GSGHs) using TEM. The magnified TEM image reveals the presence of numerous nanopores within individual nanosheets. Figure D further demonstrates that small Au NPs have been assembled onto the surface of the graphene-mesoporous silica hybrids [34]. High-resolution TEM (HRTEM) determines the number of layers, thickness, folds, and cross-sectional view of graphene at different locations [32]. In the Cu-graphene multilayer composite, 3-4 layers of graphene were observed by HRTEM imaging (Figure 1 left) [35]

Atomic force microscopy (AFM) is a high-resolution scanning probe microscope that differs from other microscopes that use electron beams or photon beams to view samples. Instead, it employs a cantilever probe that “touches” the sample during scanning. The interaction force between the cantilever and the detected surface enables it to possess 3D imaging capabilities [36,37]. During the scanning process, AFM produces images with a vertical resolution (z) of 0.02 nm and a lateral resolution (x, y) of 0.1 nm. It can be used to determine the thickness, adhesion strength, mechanical properties, topographic imaging, surface roughness, force measurements, and structural/morphological characteristics of graphene materials. Based on the nature of tip movement and tip-sample interaction, AFM can be classified into contact mode, tapping mode, and non-contact mode [37]. Tapping mode is currently the most widely used AFM imaging mode. AFM is a commonly used technique for measuring the thickness of graphene [38,39]. AFM analyses in Figure 2 revealed the height increases in BSA-GO/RGO nanosheets as compared to pristine graphene (<1.0 nm) [40]. However, the interaction between the scanning tip and the ripples on graphene can affect the measurement of graphene thickness, which requires further investigation [38,41]. Nanoscale structural variations (grain domains, grain boundaries, lattice defects, dopants, etc.) in the graphene lattice determine its application performance in electronic devices. AFM technology is also suitable for characterizing the correlation between the nanoscale structure of graphene and its electrical properties [42,43,44].

Raman spectroscopy determines the presence and structure of graphene by measuring the energy of photons scattered by the sample. It is a relatively low-cost and non-destructive characterization method. This technique is particularly suitable for characterizing carbon-based materials [45], as Raman spectroscopy can identify graphene sheets and obtain information on the number of layers, as well as analyze defects, crystal structure, and other properties of graphene [46,47]. The most intense and widely studied Raman bands in sp² carbon materials and their composites are the D band, G band, and 2D band (also known as the G’ band). The G band, which is a prominent feature of all sp²-bonded carbon materials, appears near 1580 cm⁻¹ and is usually the most outstanding spectral characteristic [48]. It originates from the stretching of C–C bonds in the lattice [48]. D band appears near 1350 cm⁻¹ and is most commonly observed in the vicinity of grain boundaries/lattice defects, such as point defects arising from vacancies, or planar defects like dislocations and stacking faults [49]. The ratio of the intensities of the D band and the G band, I_D/I_G, is often used as a measure of disorder [50]. A higher defect intensity leads to elastic scattering, and samples with high I_D/I_G values exhibit greater disorder in their graphite structure [51,52]. The 2D band, which appears near 2700 cm⁻¹, provides information about the graphene layers and their stacking [53,54]. An asymmetric and sharp 2D band with a full width at half maximum (FWHM) of approximately 30 cm⁻¹ can serve as an indicator for monolayer graphene [55]. An increase in the number of graphene layers in the sample shifts the peak to higher frequencies [56].

Under the excitation wavelength of 488 nanometers, the Raman bands of pristine graphene grown by chemical vapor deposition (CVD) can clearly observe the G band and 2D band of graphene. When graphene contains defects, such as vacancies, impurity atoms, or lattice distortions, the D band and D’ band will appear. The intensity ratio between the G band and 2D band changes with the increase in the number of layers, a characteristic that allows us to estimate the number of graphene layers through Raman spectroscopy (Figure 3).

X-ray diffraction (XRD) determines the crystalline structure and orientation of graphene by measuring the diffraction of incident X-rays on the sample [59]. XRD excels in the analysis of graphene’s crystalline structure, capable of accurately measuring key parameters such as the lattice constants and crystal orientation of graphene [60]. XRD test results reveal marginal changes in 2θ and interlayer spacing (d), and the interlayer distance between atomic planes can be calculated using Bragg’s equation. The XRD patterns of graphite and graphene exhibit distinct peaks, which can be used to distinguish the structures of graphite and graphene or to confirm the reduction in GO [60,61]. The diffraction peak appearing near 2θ = 26.3° in the XRD pattern corresponds to the (002) plane of graphene, serving as a characteristic peak for graphene (Figure 4) [62]. When graphite undergoes deep oxidation through the modified Hummers method, changes occur in its XRD pattern. A sharp peak near 2θ = 10.93° corresponds to the (002) reflection of GO [63], indicating the formation of GO and also suggesting an increase in the interlayer distance between adjacent graphene layers in graphite oxide due to oxidation [64]. XRD is not suitable for the study of single-layer graphene or GO. Reports presenting XRD patterns of graphitic materials actually report on solids where stacking with higher or lower crystallinity has occurred, depending on the width and position of the diffraction peaks [65].

Ultraviolet-visible spectroscopy can be used for the qualitative analysis of graphene. By comparing the UV spectrum of the obtained product with the UV spectrum of graphene or its derivatives, one can determine whether the obtained product is graphene or one of its derivatives. Pristine graphene and single-layer graphene oxide (GO) exhibit absorption at 262 nm and 230 nm, respectively, in the ultraviolet-visible spectrum [67]. The number of layers and the thickness of graphene can be studied through ultraviolet transmittance. As the number of graphene layers increases, the transmittance of graphene decreases from 94.3% for bilayer graphene to 83% for six-layer graphene at the same wavelength [39]. Therefore, ultraviolet-visible spectroscopy can be used to determine the number of graphene layers. According to Lambert–Beer’s Law, by establishing a curve of absorbance versus graphene concentration, the concentration of graphene can be accurately calculated by measuring its absorbance. When detecting graphene using UV-visible spectroscopy, the analysis is typically conducted in a liquid state. This is because dissolving or dispersing the graphene sample in a solvent can achieve a more uniform dispersion, thereby enhancing the accuracy and reproducibility of the measurements. Water, ethanol, and dimethylformamide (DMF) are commonly used solvents for graphene and its derivatives [68,69]. These solvents have minimal impact on the absorption spectrum of graphene, thus improving the accuracy and precision of the measurements. Additionally, ultraviolet-visible spectroscopy can also be employed to assess the dispersion degree of graphene structures (Figure 5) [70,71,72].

Fourier transform infrared spectroscopy (FTIR) reveals the formation and modification of oxygen-containing functional groups and C=C bonds [73]. The characteristic peak appearing near 1620 cm⁻¹ is typically associated with the vibration of carbon-carbon double bonds in graphene, which is a typical feature of graphene structures [74,75]. Through FTIR, we can observe the characteristic peaks of specific chemical bonds or functional groups in graphene and its derivatives, such as hydroxyl groups (-OH), carbonyl groups (C=O), carbon–carbon double bonds (C=C), as well as epoxy groups (C-O-C) or alkoxy groups (C-O) [70,76]. This allows for structural characterization of the materials. The presence and intensity of these characteristic peaks can reflect the degree of oxidation, reduction state, and surface modification of graphene [77].

2.2. Methods for Tracking Graphene Biological Exposure

The characterization methods mentioned above are frequently employed in the characterization and detection of graphene materials during their preparation or procurement. For studying the biological exposure of graphene, radioisotope tracing, fluorescein labeling techniques, and TEM are commonly used to track the accumulation and trajectory of graphene within organisms.

The carbon-14 isotope labeling method initially involves synthesizing C14-labeled GD, followed by dispersing it in a solution or pure water to prepare a suspension using probe sonication. Subsequently, biological uptake experiments are conducted. Small aquatic organisms are generally cultured in a suspension containing the added C14 label, while mice typically ingest it through tracheal administration or intravenous injection. By directly adding the C14-labeled GD suspension into the scintillation fluid (such as Gold Star) and subsequently performing scintillation counting in a Liquid Scintillation Counter (LSC), the radioactivity of the purified graphene solution can be quantitatively measured. For biological samples, after undergoing processes such as drying, the whole organism or various tissues can be directly added to the scintillation fluid for LSC counting. Alternatively, tissues can be combusted into ¹⁴CO₂ using a pyrolysis system, captured by an alkaline scintillation fluid, and then analyzed using an LSC. In addition to using an LSC, whole-body imaging and tissue sectioning of the organism can also be conducted with a high-performance autoradiography imager. Which allowed real-time radioactive imaging through direct β-particle counting and absolute radioactivity quantification was used to measure radioactivity in dried whole-body sections. The biological uptake experiment using fluorescence labeling is similar with the C14 labeling method, with the key difference being the conjugation of fluorescent dyes with GD, followed by imaging and quantitative analysis of the labeled samples using equipment such as fluorescence microscopes or flow cytometers. This allows for the understanding of the distribution and quantity changes in graphene within biological organisms.

The Carbon-14 isotope labeling method is useful for studying the accumulation and distribution of graphene in organisms such as zebrafish [78], Daphnia magna [79], algae [80], loaches [81], mice [82,83,84], and others [85,86,87]. The embryonic development of zebrafish is a common model for testing the developmental toxicity of chemicals. In studies involving zebrafish, MWCNTs (Multi-Walled Carbon Nanotubes) labeled with C14 primarily accumulated in the intestines of the fish studied, and minimal internalized radioactivity was detected in the blood and muscle tissues of exposed fish, hinting at the potential for biomagnification of MWCNTs in the food chain. Smaller, lateral-sized (25–75 nm) ¹⁴C-labeled few-layer graphene (FLG) particles exhibited greater bioaccumulation and stronger chorion penetration capabilities in zebrafish embryos [88]. The offspring exhibited more severe neurotoxicity compared to the parent zebrafish. After tracheal administration of ¹⁴C-FLG flakes to mice, intense radioactivity was observed in lung cells, indicating the internalization of 14C-FLG particles and their long-term accumulation in the lungs for over a year. In cases of acute exposure, the persistence of FLG was even higher [82]. Following intravenous injection of 14C-FLG with different lateral sizes into mice, it was found that few-layer graphene primarily accumulated in the liver. Larger graphene particles could be degraded into ¹⁴CO2 by Kupffer cells, and based on these results, a mechanism for graphene transformation was proposed [83]. After intravenous injection of Carbon-14 labeled graphene oxide (GO), quantitative assessment using a β-imager showed higher GO content in the liver of mice compared to the lungs, spleen, and kidneys [84].

Fluorescein labeling technology can also be used to trace and detect the distribution of graphene in organisms. The principle involves binding fluorescent dyes to graphene so that the graphene emits fluorescence under excitation by light of a specific wavelength. In studies tracing GO in zebrafish, the zebrafish were exposed to GO labeled with fluorescein isothiocyanate (FITC), and after incubation in the dark, fluorescence microscopy was used to study the fluorescence in the zebrafish [89]. The fluorescence of GO was mainly observed near the fish’s mouth, yolk sac, heart sections, and tail blood, indicating that GO was primarily located in these regions. To observe the interaction between GO (Graphene Oxide) and human normal lung epithelial cells (BEAS-2B), GO was labeled with fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA). After treating the cells for 24 h and staining the cytoskeleton and cell nuclei, confocal laser scanning microscopy (CLSM) was used to observe the GO nanosheets. The results indicated that most of the GO nanosheets were distributed on the cell membrane rather than inside the cells as previously reported [90]. This suggests that GO nanosheets can adsorb onto lung bronchial epithelial cells and have active interactions with them [91].

After processing biological tissues, such as sectioning, transmission electron microscopy (TEM) can also be used to directly observe graphene materials within organisms. Evidence of cellular ingestion of GO was found through TEM analysis, revealing that BeWo cells (human trophoblast cell line BeWo b30 Aberdeen) even absorbed large micro-sized GO [92]. TEM and Raman spectroscopy observations of tissue sections from both parent fish and their offspring showed that GO transferred from water to the brains of both parent and offspring zebrafish. The offspring exhibited more severe neurotoxicity compared to the parent zebrafish [93].

There is limited research on tracing the trajectory and conducting quantitative studies of graphene within organisms, especially in humans. The verification of graphene’s distribution and behavior within living organisms is typically conducted in animal models or whole organisms (e.g., zebrafish embryos and mice). Radioactive labeling is the primary quantitative method in graphene research, known for its strong tracking ability, accurate quantification, and broad applicability; however, it is limited by its complexity and high cost. Fluorescent labeling, while offering good visualization and high sensitivity, presents challenges in quantification and has limited penetration in deep tissues. Therefore, there is a need to develop more sensitive, convenient, and specific in vivo tracking and detection methods to better monitor the ingestion and distribution of graphene materials in the human body.

3. Properties, Applications, and Potential Uses of GDs

Graphene is a two-dimensional crystal made up of a single layer of carbon atoms that are bonded to each other via SP2 hybridization. The carbon atoms are arranged in a honeycomb lattice structure, with each carbon atom bonded to three others via σ bonds. The remaining π electrons of each carbon atom form delocalized π bonds with the π electrons of other carbon atoms, allowing electrons to move freely within this region and move freely within the crystal lattice without scattering, giving it excellent electronic transport properties. Because each carbon atom in graphene forms a very strong σ bond with its three adjacent carbon atoms, it exhibits excellent mechanical properties. In materials science, the high strength and toughness of graphene make it an ideal additive for manufacturing new composite materials [94,95]. Combining graphene with materials such as metals and plastics can significantly improve the mechanical properties of the material, creating stronger and more durable products. Due to its intriguing intrinsic mechanical and functional properties, graphene has attracted extensive attention in metal matrix composites as a novel ideal enforcement [96]. As a carrier in composite materials, graphene has the advantages of reducing nanoparticle agglomeration, limiting re-stacking, increasing surface area, improving mechanical strength, inhibiting leaching, inhibiting corrosion, acting as a photosensitizer, etc., so graphene-based composite materials have the advantages of easy functionalization, good biocompatibility, and great water dispersion, and become one of the best materials for preparing composite photocatalysts [94]. The hardness of CuCr25/graphene alloy prepared by sintering at 600 °C and 300 MPa increased by 11.3%, and the conductivity decreased by 4.2% compared with the CuCr25 alloy prepared by the same process without adding graphene. Compared with the CuCr25 alloy prepared by traditional process, the conductivity and hardness of the CUCR25 alloy are increased by 35% and 48%, respectively, indicating that the improvement of composition and process contributes to the improvement of performance [97]. However, most studies have assumed that graphene films are rigid, ignoring the effects of their inherent flexibility [98]. The small mechanical strain of graphene cannot meet the need for large deformation in most flexible and stretchable device applications. Kirigami is one of the effective technologies to increase the flexibility of graphene under deformation [99].

Graphene’s applications extend to our homes as well. In the form of coatings, graphene can be applied to walls and other surfaces to enhance their durability and antibacterial properties. These coatings not only protect against wear and tear but also inhibit the growth of bacteria, contributing to a healthier living environment [100]. Additionally, graphene-based paints can improve the insulation of buildings, reducing energy consumption for heating and cooling [101].

Under visible light, the synthesized TiO₂ nanorods decorated graphene sheets showed unprecedented photodecomposition efficiency, and this major photocatalytic activity was due to the synergistic effect of delayed charge recombination rate due to graphene’s high electron mobility and increased surface area due to nanoscale TiO₂ nanorods [102]. In the process of photocatalysis, the photoactive materials in the graphene-based composites, such as metals, metal oxides, semiconductors, organic matter, etc., are used as light collectors to catalyze the degradation of organic matter, and graphene is a cocatalyst to improve the catalytic performance of the composite [103]. The energy sector is also the stage for graphene to play a big role. A graphene-core radially arranged tungsten disulfide sheath is prepared by a one-step hydrothermal process. The graphene core ensures that the fibers are highly conductive, while the sheath nanosheet array creates a large number of active sites and enables rapid ion diffusion [104]. Graphene supercapacitors have extremely high power density and fast charge and discharge capabilities, which can store and release a large amount of energy in a short period of time, providing more efficient energy solutions for electric vehicles, mobile devices, etc. In addition, graphene can also be used to improve the performance of lithium-ion batteries, increasing the capacity and cycle life of batteries. Due to its excellent electrical and thermal conductivity, graphene is expected to replace traditional silicon materials for the manufacture of smaller, faster, and more energy-efficient electronic components. Graphene transistors, for example, operate much faster than silicon transistors and can greatly increase the processing power of computers [95].

The high electron mobility, large specific surface area, high mechanical rigidity, high thermal and optical properties, and biocompatibility of graphene and its derivatives make them reasonable candidates for use in various fields, especially water purification [102,105]. The high electronic conductivity of graphene leads to easy transfer and separation of charges, which is useful for delaying charge recombination. The high surface volume ratio of graphene’s two-dimensional planar structure creates a large specific surface area that increases the reaction site [102]. Graphene exhibits a high sensitivity to perturbations in the electron charge distribution generated when the target substance is adsorbed on its surface, thus altering the sensing response of graphene-based resistive gas sensors [95,106]. A single layer of crumpled graphene could be used to further improve the sensitivity of graphene-based devices. Crumpling causes a change in the wettability of the device because the solvent interacts differently with the surface composed of ripples. Thus, this provides opportunities for the functionalization of graphene layers, fabrication of devices, and fine-tuning of doping strategies [106]. Graphene also has important applications in biomedicine. It can be used to manufacture biosensors to achieve high-sensitivity detection of biomolecules. Another advantage of graphene is its low impact on the environment, making it more suitable for sensing purposes than other metals [4]. Graphene not only can compress but also can distinguish between small pressure changes, so graphene has potential applications for pressure sensors [95,107].

In addition to excellent conductivity and mechanical properties, graphene also has unique optical properties, with high mobility and optical transparency. Excellent optical properties make graphene a promising material for high-performance optoelectronic devices. The transmittance of single-layer graphene in the visible light region is more than 97%. These properties make graphene the most promising and exciting material, which can have a wide range of application prospects in the important fields of nanodevices and sensors [108,109].

The application field of graphene continues to expand and deepen, and its unique properties provide new ideas and methods for solving many challenges faced by modern science and technology (Figure 6). With the continuous progress of graphene preparation technology and the reduction in costs, graphene batteries are expected to replace traditional lithium-ion batteries in the future, providing longer endurance for electric vehicles, wearable devices, etc. The flexibility of graphene allows it to be made into electronic devices of various shapes, such as flexible screens, wearable devices, and so on [110]. However, the high preparation cost and complex preparation process of graphene limit its large-scale production and application. Although significant progress has been made in the preparation of graphene in recent years, further efforts are needed to achieve large-scale production.

The future prospects of graphene are largely increasing, and its application areas are expected to continue to expand, and the market size will continue to expand. In terms of technological innovation, the preparation technology of graphene is the key to its large-scale commercial application. At present, the preparation technology, such as the REDOX method and chemical vapor deposition (CVD), has been continuously optimized, which has improved the preparation efficiency and product quality. Heteroatomically doped graphene and the preparation of graphene heterostructures are two methods to overcome the zero bandgap and two-dimensional (2D) nature of graphene. With the continuous deepening of research and continuous progress of technology, it is believed that graphene will play a key role in more fields and promote the leapfrog development of modern science and technology.

4. Human Exposure Pathways to GDs and Their Health Risks

Various exposure ways to graphene and its derivatives (GDs) have been investigated, including inhalation, ingestion, dermal exposure, etc., which pose potential threats to organisms, like organ damage, skin irritation, and potential cell damage, etc. The study methods, types of graphene, tested objectives, exposed dose, and identified health risks of previous studies are listed in

Table 1. A summary of previous studies on exposure to graphene-based materials.

Exposure Pathways	Experimental Types	Studied Objective	Material	Dose	Exposure Time	Effects	References
Inhalation	In vivo intranasal instillation	mice	Graphene quantum dots	0.1 or 1 mg/kg weight	28 d	dysfunction, histopathological alternations, and fibrosis in the lung, liver and kidney tissues of mice; disruption of the iron balance and redox equilibrium of these respiratory organs.	[15]
	In vivo Intratracheal instillation	mice	Graphene oxide	3.24 μg/mouse	1, 3, 28, 90 d	Transcriptomic changes associated with pulmonary inflammation and increased risks of atherosclerosis development and fibrosis in the lung and liver.	[112]
	In vivo Intratracheal administration	mice	14C-few-layer graphene (14C-FLG) flakes	0.17 mg per kg b.w.	repeated once a week for 4 weeks	Material accumulation in the lungs after one-year post-exposure	[82]
	In vivo Nostril instillation	mice	GO	50 μL dispersions of 1mg/mL GO	1, 7, 28, 90 d	Granulomas in lungs persisting for up to 90 days	[113]
	In vivo Water exposure	oyster	Graphene oxide	2.5, 5 mg/L	14 d	Elevated lipid peroxidation and changes in glutathione-s-transferase (GST) activities in gills and digestive gland tissues.	[114]
	In vivo Water exposure	oyster	Graphene oxide	1, 10 mg/L	72 h	Elevated lipid peroxidation, loss of mucous cells, hemocytic infiltration, and vacuolation in gills.	[115]
	In vitro	A549 epithelial cells of the human lung	Synthesized GDs	0.1–1000 µg/mL	24, 48, 72 h	reduced cell viability at high concentrations	[116]
	In vitro	BEAS-2B, Human normal lung epithelial cells	Graphene oxide	1–100 μg/mL	24 h	suppress the efflux function of ATP-binding cassette transporters	[91]
Ingestion	In vivo Water exposure	oyster	Graphene oxide	1, 10 mg/L	72 h	reduced total protein levels in digestive gland tissues	[115]
	In vivo Water exposure	oyster	Graphene oxide	2.5, 5 mg/L	14 d	Elevated lipid peroxidation and changes in glutathione-s-transferase (GST) activities in gills and digestive gland tissues.	[114]
	In vivo oro-pharyngeal aspiration	mice	GO sheet	1, 10 μg	Once per 14 days (lasting for 84 days)	Long-term DNA double-strand breaks at a higher dose of micrometric GO sheets	[11]
	In vivo Oral administration	mice	rGO nanosheets	60 mg/kg body weight/d	5 d	A short-term decrease in locomotor activity and neuromuscular coordination	[10]
	In vitro	Caco-2 cells derived from human colorectal adenocarcinoma	GOs and graphene nanoplatelets	0–80 μg/mL in culture medium	24, 48 h	ROS formation after GO exposure; low acute toxicity at high concentrations of graphene nanoplatelets	[117]
Dermal exposure	In vitro	human skin HaCaT cell	layered graphene (FLG), GOs (GO1, GO2, GO3)	FLG (0.005 to 90 μg/mL culture medium) GO (0.005 to 100 μg/mL culture medium)	72 h	Impaired mitochondrial activity associated with plasma membrane damage	[16]
	In vitro	Human skin fibroblast cells (CRL-2522)	graphene oxide (GO) and graphene sheets (GS) of various sizes and oxygen content	3.125–200 μg/mL	1 h	cytotoxicity, and more reactive oxygen species (ROS) formation	[118]
	In vivo intradermal GBN injection	Growing feathers (GF) of chickens	oxygen-functionalized graphene nanomaterial (f-GNB)	60 g/chicken	0, 0.25, 1, 2, 3, 4, 5, and 7 days	inflammatory immune response activity initiated by cellular/tissue damage	[119]
Intraperitoneal and intravenous administration	In vivo Intraperitoneal and intravenous administration	Healthy albino mice	PEGylated reduced graphene oxide (PrGO)	10 mg/kg body weight	3, 7, 14 and 21 days	PrGO was distributed in the brain, liver, kidney, spleen, and bone marrow and might induce inflammatory responses; Cross blood–brain barrier; organ damage.	[120]
	in vivo tail vein injection	mice	Graphene nanosheets	1 mg/kg body weight	1 or 7 days	Th2 immune responses which may induce adverse allergic reactions.	[121]

.

4.1. Inhalation

Inhalation is the primary exposure pathway to airborne graphene particles. The release of airborne graphene nanoparticles in the graphene-based manufacturing industry has been identified, raising the health risks of workers who are the first target population in the industry [122]. In aerosolized or powder forms, the graphene-based materials are easier to deposit into the deep lung area [123]. Thus, it is highly necessary to study the health risks of inhalation exposure to graphene-based materials. Intranasal instillation, intratracheal administration, and in vitro cell-based experiments are often used to simulate the respiratory exposure of GDs.

Inhalation exposure to GDs could enhance the dysfunction, fibrosis, inflammation, granuloma formation, and cell damage of respiratory organs according to animal-based experiments. The research in which mice were intranasally administered with different levels of graphene quantum dots (GQDs) (0.1 or 1 mg/kg weight) found dysfunction, histopathological alternations, and fibrosis in the lung, liver, and kidney tissues of mice. In addition, the graphene quantum dots disrupted the iron balance and redox equilibrium of these respiratory organs [15]. Smad3 (one type of protein in the Smad family), the phosphorylation of Smad3, and the nuclear translocation of p-Smad3 are essential indicators of fibrogenesis [124,125]. Immunohistochemistry (IHC) staining was applied to measure the expression levels of phosphorylated Smad3 (p-Smad3) in the lung, liver, and kidney tissues of mice. Exposure to BW N-GQDs and A-GQDs1 mg/kg may increase the expression levels of p-Smad3 in lung and liver tissues to some degree (Figure 7a,c). In addition, Western blotting (WB) analysis was also used for detecting the expression levels of proteins (Smad3, p-Smad3, and TGF-ß1). The enhancement of the p-Smad3 protein expression levels in lung and liver tissues and the increased expression levels of TGF-ß1 in lung, liver, and kidney were observed (Figure 7d,e). These observations proved the induced fibrosis in the lungs, liver, and kidney by exposure to GODs. Transcriptomic changes associated with pulmonary inflammation and increased risks of atherosclerosis development and fibrosis in the lung and liver were identified by GO intratracheal instillation (54 μL/mouse) [112]. Long-term accumulation of the GDs was observed in the lungs, both in repeated low-dose and acute high-dose exposure of ¹⁴C-few-layer graphene (¹⁴C-FLG) flakes in mice by tracheal administration, even after one year since post-exposure [82]. An experiment of nostril instillation on mice found that GO exposure could lead to granuloma formation lasting for 90 days, which might induce pulmonary inflammation [113]. The oysters were placed in chambers containing seawater with various concentrations of GO. Gills were analyzed to explore the effects of graphene inhalation exposure. Elevated lipid peroxidation and changes in glutathione-s-transferase (GST) activities were induced by short-term GO exposure (2.5–5 mg/L) [114]. Short-term exposure to 10 mg/L can lead to elevated lipid peroxidation, loss of mucous cells, hemocytic infiltration, and vacuolation in gills [115].

Exposure to GDs could also reduce the cell vitality and accumulate more toxic substances of lung epithelial cells from in vitro experiments. A study focusing on the A549 epithelial cells of the human lung cultivated in the culture medium with 0.1–1000 µg/mL of GD found that longer exposure to the GDs of higher concentrations could induce more cytotoxicity in the lung cell and reduce the cell vitality [116]. And 24 h exposure to GO could suppress the efflux function of ATP-binding cassette transporters on the cytomembrane of BEAS-2B, human normal lung epithelial cells, which could hinder the transfer of toxic matter out of the cells, especially toxic metal ions, which could induce a series of toxic reactions through oxidative stress response and accelerated cell death [91].

4.2. Ingestion

As graphene-based materials are eligible substances as drug carriers nowadays, ingestion is also an important exposure pathway to GDs, given that most patients prefer oral therapy during the disease treatment [10,126,127]. Oral administration is often applied to simulate the ingestion exposure to GDs.

Exposure to GDs could induce DNA damage, reduced locomotor activity and neuromuscular coordination, and declined protein level and oxidation stress in digestive gland tissues of animals. From in vivo studies, exposure to a high level of rGO (60 mg/kg body weight/d) nanosheets (60 mg/kg body weight) for 5 days via oral administration caused a short-term decrease in locomotor activity and neuromuscular coordination of mice [10]. The rotarod test is applied to the mice with 2-day post-exposure in order to analyze potential neurotoxicity, motor coordination, muscle tone, and balance in mice [128]. The longer time mice spent on the rotarod represents the less physical decline and neuromuscular coordination damage. After 2 days since the final treatment, mice exposed to HEPES buffer and chow (control groups) performed better than mice treated with rGO in the four days of training (Figure 8A), who displayed poor balance and often fell down. After 16 days since the final administration, mice in control groups spent similar time on the rotarod with the rGO-treated mice in the initial 2 days of training, while the rGO-treated mice spent less time on the rotarod in the third and fourth days of training, compared with control groups (Figure 8B). It is obvious that large rGO-treated mice spent less average time on the rotarod than mice exposed to small rGO on the fourth day of training since 2, 16, and 61 days after the final treatment (Figure 8). These results are indicative of the physical decline and decreased neuromuscular coordination of mice exposed to a high dose of rGO. Long-term DNA double-strand breaks were observed after repeated oropharyngeal aspirations of a high dose of micrometric GO sheets (10 μg/mouse) [11]. The oysters obtained elevated lipid peroxidation and changes in glutathione-s-transferase (GST) activities in digestive gland tissues after 14-day GO exposure (2.5–5 mg/L), which are indicators of oxidative stress and detoxification processes in organisms [114]. And 72 h exposure of 5-7 mg/L GO can lead to reduced total protein levels in digestive gland tissues [115].

Ingestion exposure to GDs could induce oxidative stress leading to cellular damage and cause acute toxicity based on in vitro studies. A study focused on the Caco-2 cell line, which is derived from human colon adenocarcinoma and widely applied in pharmaceutical research as one of the gold-standards for in vitro studies about the intestinal tract. The cells were exposed to 4 kinds of GO and graphene nanoplatelets (GNP) (0–80 μg/mL in culture medium) for 24 h and 48 h. There was reactive oxygen species (ROS) formation after GO and GNP exposure, which could cause oxidative stress, leading to cellular damage. And graphene nanoplatelets at higher concentrations caused low acute toxicity to the intestinal cells [117].

4.3. Dermal Exposure

Dermal exposure is one of the common exposures to GDs because skin is the major barrier between the human body and the environment, especially for workers in the graphene-based manufacturing industry and customers who use graphene-based applications. Previous studies have found that cutaneous contact with graphene has been associated with skin disorders, like contact dermatitis, carbon fiber dermatitis, hyperkeratosis, and naevi [129,130]. In vitro experiments are primarily applied to simulate the skin exposure to graphene.

Dermal exposure might cause reduced cell vitality, cell damage, ROS formation, and exert cytotoxicity according to in vitro studies. ROS are highly reactive molecules that can damage cellular components like proteins, lipids, and DNA. This oxidative stress contributes to cellular damage, inflammation, and the development of various diseases, including cancer [131]. HaCaT keratinocytes, human skin cells, were applied to be exposed to the GDs (0.005–100 μg/mL culture medium) with different levels of oxidation. And the cells were analyzed every 24 h. The results showed that high-level GO3, the largest and most oxidized compound, presented the highest cytotoxicity and induced mitochondrial and plasma-membrane damages with EC_50s of 5.4 and 2.9 μg/mL, respectively. FLG, the less oxidized compound, showed less cytotoxicity and damaged mitochondria and plasma-membrane damages at higher concentrations with EC_50s of 62.8 μg/mL and 45.5 μg/mL, respectively [16]. Longer-term exposure to FLG and GO3 at higher concentrations could result in more reduced mitochondrial activity and more cytotoxic effects at the skin level (Figure 9). Another in vitro study focusing on human skin fibroblast cells (CRL-2522) exposed to GO and GS found that GO and GS showed cytotoxicity and generated more reactive oxygen species (ROS) in human skin fibroblast cells. While aggregated GS particles are more cytotoxic than reversibly aggregated GO and generated more ROS in human skin fibroblast cells than reversibly aggregated GO [118].

Inflammatory immune response activity initiated by cellular/tissue damage was found from in vivo study, which could lead to the release of pro-inflammatory cytokines and other molecules. This immune activation, while initially protective, can result in chronic inflammation if graphene particles accumulate in tissues over time [132]. An in vivo study of dermal exposure to GDs analyzed the growing feather dermis of chickens injected with the oxygen-functionalized graphene-based nanomaterials (f-GNB) (60 μg/chicken) to explore the local and systemic cellular/tissue response to GBN. The reason chicken was chosen as a tested animal is because the chicken has a highly evolved immune system similar to the human immune system, as an accepted animal model for biomedical research [133,134]. Also, the growing feather, as a skin derivative, was used as a dermal test-site over the skin because it is easy to conduct intradermal injection, the location of the injection site is predictable, and the size of the biopsy sample is uniform, which causes little pain to the chicken as well. Higher and sustained leukocyte infiltration/presence of mononuclear leukocytes (lymphocytes and macrophages) were observed in injected growing feathers throughout the 7-day post-injection period, which were the indicators of inflammatory immune response activity induced by cellular/tissue damage caused by injection of f-GBN.

4.4. Intraperitoneal and Intravenous Administration

Intraperitoneal and intravenous administration are related to intentional exposure to nanomaterials designed for biomedical applications because GFMs have been used in the drug production, for example, as a carrier for drug delivery [126,127].

The intraperitoneally and intravenously administrated GDs could spread through blood and major organs, which might induce blood–brain barrier crossing and organ inflammatory responses and damage. The blood–brain barrier is a protective membrane that restricts the passage of harmful substances into the brain. Once inside the brain, graphene can cause neurotoxicity, neuroinflammation, and oxidative stress, potentially leading to cognitive decline, neuronal damage, or even brain disorders [135]. In vivo studies, healthy albino mice were both intraperitoneally and intravenously administrated with 10 mg/kg body weight of PrGO. Their major organs, including the liver, brain, kidney, and spleen, were collected for further analysis at the end of 3, 7, 14, and 21 days of post-exposure. After intravenous administration, PrGO nanoparticles quickly transfer throughout the circulatory system and are subsequently translocated to various organs, including the brain, liver, kidney, spleen, and bone marrow of mice. After intraperitoneal administration, the PrGO nanoparticles are gradually absorbed by the abdominal cavity and are slowly translocated and accumulated in major organs (Figure 10). The presence of PrGO in major organs potentially suggested the possibility of cross blood–brain barrier and organ inflammatory responses and damage, especially congestion in the kidney, acute liver injury, and increased splenocyte proliferation after repeated administration of PrGO [120]. Another in vivo study investigated the influences of exposure to graphene nanosheets (1 mg/kg body weight) on mice with tail vein administration. Exposure to nanosheets induced a Th2 immune response 1 day post-injection in the lungs of mice, which consisted of neutrophilic influx and a significant increase in interleukin (IL)-5, IL-13, IL-33, and its soluble receptor (sST2) in the bronchoalveolar lavage fluid. It is speculated that the use of graphene nanosheets as nanocarriers for drug delivery might lead to Th2 immune responses that may aggravate adverse allergic reactions [121].

Based on the in vivo studies whose animal models have similar physiological characteristics with humans and the in vitro studies that used human cells, the potential risks to human beings of several exposure pathways to GDs are summarized and illustrated in Figure 11. GDs could transfer to the circular system of people and accumulate in blood, cells, and major organs, including the lung, liver, kidney, and spleen. GDs exposure could induce organ and cell inflammatory responses and damage, such as pulmonary edema granuloma formation, decreased aspartate aminotransferase level, disrupted kidney function, declined cell vitality, cytotoxicity, increased cell apoptosis, etc. [136]. These changes at the organ and cell levels might lead to adverse tangible influences on people. For example, the exposure to GDs could decrease the locomotor activity and accelerate the aging process of people [10]. GDs exposed to the female even lead to abnormal development of the offspring [137]. Above all, the potential health risks to people of GDs exposure should be paid more attention to investigate the quantitative influences of GDs on humans.

4.5. Other Exposures Routes to Graphene

Graphene is a suitable candidate for many applications in the food industry, from food packaging to agriculture. In recent years, the application of graphene oxide in the electronics, machinery, optics, and food and clothing manufacturing industries has become more and more extensive, making the human body’s exposure to graphene oxide greatly increased [138,139,140], and there are many ways of exposure, including inhalation through the lung in occupational places, oral ingestion through biological enrichment in water and soil, and oral and intravenous injection in the biomedical field [141]. Through inhalation, people are constantly exposed to environmental and occupational irritants that can cause adverse health effects, such as irritation-induced asthma [142]. Graphene, when bound/adhered to epithelial tissue, can stimulate mRNA expression of genes involved in cell proliferation and growth [143]. GO causes up-regulated expression of genes and proteins related to stress, apoptosis, inflammation, immunity, antioxidants, endocytosis, and transcription, and down-regulated expression of genes and proteins related to development and metabolism. Gastrointestinal exposure to GO can induce the decrease in flora species abundance and dysregulation of community structure in non-pregnant mice and zebrafish, suggesting that GO exposure during pregnancy may destroy maternal intestinal flora [141,144]. Focusing on the biological effects of graphene oxide (GO) and reduced graphene oxide (rGO) materials on PC12 cells, a traditional nerve cell line, found that GO and rGO produced significant toxic effects on PC12 cells in a dose–and time-dependent manner. Moreover, apoptosis appears to be a response to toxicity [145]. Graphene-based materials show potential applications in dentistry due to their excellent physicochemical properties. However, the use of graphene and its derivatives increases their risk of exposure to periodontal cells. Human periodontal ligament cells (hPDLCs) were isolated, and the cytotoxic behavior and related signaling pathways of hPDLCs damaged by graphene oxide (GO) nanoparticles were studied. The results showed that the cytotoxicity of GO to hPDLCs came from the coating of GO nanosheets on the membrane surface, which blocked the phosphorylation of epidermal growth factor receptor on the membrane [146]. Graphene exposure caused acute lung injury in the lung tissue of the mice, with pulmonary edema, inflammatory cell infiltration, and cell damage as the main symptoms, and it was dose-dependent. After exposure by tracheal infusion, graphene is mainly deposited in the lungs and cleared from the lungs to the gastrointestinal tract by the action of mucocilia and alveolar macrophages and excreted in the stool. A small part of the deposited graphene can break through the lung Qi blood barrier, enter the blood circulation, and distribute in the liver and spleen [147].

With the increasing use of graphene in consumer products, in the production and use of graphene nanoproducts, the graphene contained in it will inevitably be released into the environment through various ways, affecting terrestrial and aquatic organisms and human health. In the environmental and health risk assessment of pollutants, the study of the exposure process is an essential link, and the accurate quantification of the exposure dose of organisms in the actual environment is an important factor affecting the reliability of the risk assessment results.

5. Comparison of Methods and Health Risks Between GDs and Dust Exposure Studies

Significant health risks induced by the exposures to GD and dust were determined. Dust exposure is generally more established as a cause of chronic diseases, while the effects of graphene on human health are still being actively researched, with more emphasis on acute toxicity and inhalation risks. Graphene exposure induces respiratory risk (lung inflammation, oxidative stress, or even fibrosis), DNA damage, cardiovascular effects (increased oxidative stress and inflammation), systemic toxicity (liver and kidney damage after prolonged exposure), skin irritation, and potential cell damage, which are mentioned in Section 4. Dust exposure is a known cause of respiratory diseases, including cancer, pulmonary fibrosis, and cardiovascular diseases because of viruses, microorganisms, and hazardous chemical components attached to dust. For desert dust, most studies focused on respiratory diseases (30.8–44.9%), cardiovascular diseases (19.6–25.0%), and all-cause diseases (9.5–15.9%). Other less-studied health conditions included infectious diseases, allergic skin and eye problems, cerebrovascular, adverse birth outcomes, allergic diseases, health-related quality of life, under-five mortality, etc. [148]. People, especially construction workers, are mostly exposed to construction dust through respiratory and dermal exposure. The most common hazardous factor in construction dust is silica. And construction dust was found to be associated with chronic obstructive pulmonary disease [149], pneumoconiosis [150], pulmonary fibrosis [151], etc. Numerous studies have shown that coal dust exposure is closely associated with decreased lung function, coal worker’s pneumoconiosis, chronic obstructive pulmonary disease, lung cancer, and cardiovascular diseases [152]. Mine dust was identified as a culprit of occupational diseases (e.g., pneumoconiosis, silicosis), respiratory diseases (e.g., chronic obstructive pulmonary disease), cancer (lung cancer), and other diseases like diabetes [21]. Road dust was proved to increase the risk of heart diseases, stroke, respiratory diseases, etc., because it is a great carrier of viruses, pathogenic microorganisms, and carcinogenic heavy metals from traffic emissions [23]. Moreover, soil dust exposure was associated with adverse health effects, such as asthma, fungal infections, and premature death [153].

So far, epidemiological studies, particularly cohort studies and cross-sectional studies, are often applied to investigate the health risk of dust exposure [151,154]. For example, to investigate whether occupational exposure to vapors, gasses, dusts, and fumes increases the risk of mortality from chronic obstructive pulmonary disease (COPD), particularly among never smokers, a study focused on a cohort of 354,718 male construction workers, of whom 196,329 were exposed to these occupational hazards. Exposure to inorganic dust, wood dust, vapors, fumes, gasses, and irritants was assessed using a job-exposure matrix, with a focus on exposures during the mid-1970s. The cohort was followed from 1971 to 2011. Relative risks (RRs) were calculated using Poisson regression models, adjusting for age, BMI, and smoking status. Among the exposed workers, there were 1,085 deaths from COPD, including 49 never smokers. Workers with any occupational exposure to vapors, gasses, fumes, and dust had an increased risk of COPD mortality (RR 1.32; 95% CI, 1.18–1.47). The proportion of COPD cases attributable to occupational exposure was 0.24 among all workers and 0.53 among never smokers. It concluded that occupational exposure to airborne pollutants (e.g., dust, gasses, etc.) increases the risk of mortality from COPD, particularly among never smokers [22].

While toxicological studies, typically through in vitro or in vivo models. are mainly used to determine the harmful effects of graphene exposure [115,155]. But some cell-based and animal-based experiments did not reflect the realistic conditions of human exposure to graphene-based materials. Experimental inhalation and ingestion exposure to graphene-based material were conducted by tracheal administration or oral administration. The exposed doses were set much higher than the realistic exposure concentrations. For example, the inhalation-exposed doses were 1 mg/kg weight for mice, 10 mg/L for oysters, and 1000 µg/mL culture medium for epithelial cells of the human lung, which are much higher than the realistic detected concentrations of GDs [15,115,116,155]. Few studies had humans as studied objects. The clinical use of GDs cannot proceed until their biosafety concerns are fully resolved [155].

Thus, GDs exposure presents novel concerns due to nanomaterial properties and relatively recent widespread usage, while dust exposure has been a long-standing public health issue, with well-established links to respiratory and systemic diseases. As the application of graphene-based materials increases, the graphene nanoparticles might become a potentially risky composition in dust, which needs further investigation.

6. Conclusions

GDs are emerging materials, presenting excellent electronic, mechanical, optical, and thermal properties, which makes graphene applied for photocatalysts, energy storage devices, sensors, and biomedical delivery. However, the increasing use of graphene and its derivatives (GDs) has significantly enhanced human exposure to these materials. Given that graphene is a novel nanoscale concern, we reviewed the properties and applications, characterization and detection methods, exposure pathways, and health risks to humans of graphene and its derivatives, which could offer references and suggestions for further graphene-related studies.

The characterization methods for graphene have become relatively well-established, including microscopy (e.g., SEM, TEM, AFM) and spectroscopic analysis (e.g., Raman spectroscopy, XRD, UV-Vis). Radioactive labeling and fluorescence methods are used for tracing GDs in vivo but cannot be considered as convenient and mature methods. Further development is needed to enhance the convenience and applicability of tracing methods.

There are various exposure pathways to GDs for humans, such as inhalation, ingestion, skin, intraperitoneal, and intravenous exposure, which exert harmful influences on human health, including organ damage (inflammation, dysfunction, fibrosis, granuloma formation, etc.) and cell damage (reduced cell vitality, cytotoxicity, etc.). At the body level, people who are exposed to GDs for a long term might suffer from reduced locomotor activity and neuromuscular coordination and an accelerated aging process. In addition, the differences in study methods and health risks between GDs and dust exposure studies were discussed.

In the future, new methods that can simulate the realistic exposure scenario need more exploration. Because many in vivo and in vitro experiments did not reflect the realistic conditions of human exposure to graphene-based materials. There are high hopes that epidemiological studies could be applied in the graphene study with more clinical and diagnosed information of patients in the future.