Surface area

Surface area plays a central role in the biological interactions of nanomaterials.Reference Nel, Madler, Velegol, Xia, Hoek, Somasundaran, Klaessig, Castranova and Thompson¹⁸ Small nanoparticles (<10 nm) have a significant fraction of their atoms exposed on their surfaces. Monolayer graphene represents an extreme case, in which every atom lies on the surface and, in fact, each atom is exposed to the surrounding medium on two sides, giving rise to the theoretical maximum specific surface area of an sp ²-hybridized carbon sheet of about 2630 m²/g. As shown in Figure 1, the surface areas of other GFNs decrease as the number of layers, N, increases according to the scaling law A/m ∼ 1/N, where A/m is the specific surface area, or total area per unit mass.

Figure 1.

Structure and property variations in graphene-family nanomaterials. (a) Number of layers determines surface area. A plot of the Brunauer–Emmett–Teller surface area (A) and vendor-specified numbers of layers (N) for commercial few-layer-graphene (FLG) samples (squares) agrees well with the simple law A = 2630/N m²/g (curve). (b) Ranges of N and lateral dimension L in various graphene materials. Solid symbols represent commercial FLG samples, and open symbols represent graphene oxide.Reference Guo, Kim, Han, Shenoy, Huang and Hurt^19–Reference Wang, Li, Hu, Lin, Li and Lin²¹ The bold black curve shows a 5-μm aerodynamic diameter (D _ae), calculated from N and L following Reference Reference Sanchez, Jachak, Hurt and Kane1, which is assumed to separate respirable and nonrespirable materials.Reference Schinwald, Murphy, Jones, Macnee and Donaldson²² Most FLGs with L > 20 μm still lie in the respirable region (D _ae< 5 μm) because they are ultrathin, but might not be cleared effectively following lung deposition. One commercial sample falls into this category.

Because of their high surface areas, surface phenomena such as physical adsorption and catalytic chemical reactions can affect the biological response to GFNs. The closest analogue to graphene is a pristine single-walled carbon nanotube (SWCNT), which is similarly hydrophobic and has a theoretical outer surface area of 1300 m²/g (although this surface area is significantly reduced in many cases to values below 500 m²/g by bundling). The high hydrophobic area of SWCNTs has been associated with the adsorption of molecular probe dyes and in vitro artifacts in toxicology assays,Reference Casey, Herzog, Davoren, Lyng, Byrne and Chambers^23, Reference Worle-Knirsch, Pulskamp and Krug²⁴ as well as the depletion of folic acid and other micronutrients from cell culture media, leading to cell growth inhibition.Reference Guo, Bussche, Buechner, Yan, Kane and Hurt²⁵ Adsorptive interferences have also been reported for carbon blackReference Monteiro-Riviere and Inman²⁶ and are expected for other materials with large hydrophobic surface areas, such as GFNs. The high surface areas of these materials make surface reactions potentially important, including deactivation of antioxidantsReference Liu, Gurel, Morris, Murray, Zhitkovich, Kane and Hurt²⁷ and production and quenching of reactive oxygen species (ROS).Reference Fenoglio, Tomatis, Lison, Muller, Fonseca, Nagy and Fubini²⁸

Number of layers

The number of graphene layers in a stack is important because it determines specific surface area and bending stiffness. Simple geometric considerations for large, thin plates give A/m = 2/ρd, where ρ is the material density and d is the thickness. In turn, d is given by N _layerd ₁, where N _layer is the number of layers and d ₁is the thickness of a single layer, which is 0.34 nm for unoxidized few-layer graphene (FLG). This yields

(1)$$A/m{\rm{ = 2/}}\rho N_{{\rm{layer}}} d_{\rm{1}} \,,$$

which shows that the specific surface area (m²/g) is inversely proportional to the number of layers (Figure 1). The adsorptive capacity for biological molecules is expected to increase significantly as the number of layers decreases. Stiffness is reported to be important in the pathological response to fibers and carbon nanotubes,Reference Poland, Duffin, Kinloch, Maynard, Wallace, Seaton, Stone, Brown, Macnee and Donaldson¹³ but its role for platelike materials is still unknown. Thin materials, such as most GFNs, are quite deformable by weak forces such as water surface tension.Reference Guo, Kim, Han, Shenoy, Huang and Hurt^19, Reference Bellido and Seminario^29, Reference Patra, Wang and Kral³⁰ In contrast, some multilayer-thick materials can act as rigid bodies during cellular interactions.

Lateral dimension

Lateral dimension has no significant effect on specific surface area (m²/g), but it is relevant for cell uptake, renal clearance, blood–brain barrier transport, and many other biological phenomena that determine the fate of particles in the body. The cellular uptake of platelike nanostructures, by either endocytosis into normal cells or phagocytosis into specialized macrophages, is poorly understood but is likely sensitive to lateral dimension, which represents the maximum dimension of the object being engulfed. The lateral sizes of GFNs span orders of magnitude (Figure 1), from 10 nm (the size of some proteins) to >20 μm (larger than some cells). Cells might thus adhere to and spread on larger GFNs, or successfully internalize smaller GFNs, or experience some form of frustrated endocytosis or phagocytosis with implications for biological risk. Sanchez et al.Reference Sanchez, Jachak, Hurt and Kane¹ reported macrophage uptake of FLG samples with lateral dimensions of 0.5 μm, 0.88 μm, and 5 μm, but not 25 μm, although even a 25-μm sample is likely respirable because of its ultrathin third dimension (Figure 1), as also reported and discussed by Schinwald et al.Reference Schinwald, Murphy, Jones, Macnee and Donaldson²²

Surface chemistry

The graphene family includes materials with widely varying surface chemistries, even before any specific biofunctionalization is carried out. Graphene oxide surfaces are partially hydrophilic (typical water contact angle of 40–50°),Reference Hasan, Rigueur, Harl, Krejci, Gonzalo-Juan, Rogers and Dickerson^31, Reference Hsieh and Chen³² are capable of hydrogen bonding and metal-ion complexing,Reference Yang, Chang, Wang, Liu, Chen, Wang, Liu and Cao³³ and contain negative charges on edge sites associated with carboxyl groups.Reference Cote, Kim and Huang³⁴ The pristine graphene surface, in contrast, is hydrophobic (water contact angle near 90°)Reference Kotsalis, Demosthenous, Walther, Kassinos and Koumoutsakos³⁵ and capable of adsorbing biomolecules or being partitioned into membranes by hydrophobic forces. Pristine graphene is capable of biochemical reactions primarily at edge or defect sites. Reduced graphene oxide is intermediate in hydrophilicity and in reactivity, as it contains vacancy defects produced during oxygen removal.Reference Bagri, Mattevi, Acik, Chabal, Chhowalla and Shenoy³⁶ Much of the biomedical work has been done on nanoscale GO because of its higher dispersibility in aqueous phases. Pristine graphenes (monolayer or FLG) are very poorly dispersible in water and require surfactants or other stabilizing agents for any meaningful application requiring suspension in biological fluids.

Purity

Unlike carbon nanotubes (CNTs), GFNs are not typically grown catalytically and do not contain residual metal catalysts. However, some GFNs might contain residual intercalants, chemical additives used to separate the layers in the bulk graphite feedstock, that were not fully removed by washing.Reference Kim, Cote and Huang³⁷ GO synthesis uses a variety of reagents that can leave soluble residues in suspension if they are not properly washed, and the washing of GO can be tedious because of gelation caused by physical interactions between the giant molecular disks.Reference Kim, Cote and Huang³⁷ The reagents used in various GFN syntheses include permanganate, nitrate, sulfate, chromate, peroxide, persulfate, and hydrazine. GO can also contain lower-molecular-weight oxidative debris that is noncovalently attached to the primary sheets,Reference Rourke, Pandey, Moore, Bates, Kinloch, Young and Wilson³⁸ and the biological response to this organic material is unknown.

Small-molecule and ion adsorption

A defining characteristic of GFNs is high surface area, which should lead to the adsorption of small-molecule solutes from physiological fluids (Figure 2b). Adsorption on carbon surfaces is generally favored for molecules with low solubility, partial hydrophobicity, or positive charge (for the common case of negatively charged graphene) and for molecules with conjugated π bonds that impart planarity and allow π–π interactions with graphenic carbon surfaces. The biological consequences might include (1) micronutrient depletion;Reference Guo, Bussche, Buechner, Yan, Kane and Hurt²⁵ (2) artifacts in assays that rely on dye-based molecular probes;Reference Casey, Herzog, Davoren, Lyng, Byrne and Chambers^23, Reference Worle-Knirsch, Pulskamp and Krug²⁴ (3) the capacity to carry small-molecule drug cargoes;Reference Yang, Wang, Huang, Ma, Huang, Yang, Duan and Chen⁴⁷ and (4) synergistic or antagonistic toxic effects when graphenes coexist with small-molecule toxicants, whose bioavailability can be increased or decreased as they partition to graphene surfaces.

Most attention on small-molecule interactions has been related to drug delivery, where functionalized nanoscale GO has been used as a carrier for small-molecule drugs,Reference Zhang, Xia, Zhao, Liu and Zhang^11, Reference Sun, Liu, Welsher, Robinson, Goodwin, Zaric and Dai^20, Reference Huang, Xu, Lin, Wang, Wang, Zhang, Zhou, Guo and Cui^40, Reference Zhang, Guo, Huang, Liu, Guo and Zhong⁴⁸ including the cancer drugs doxorubicin,Reference Zhang, Xia, Zhao, Liu and Zhang^11, Reference Sun, Liu, Welsher, Robinson, Goodwin, Zaric and Dai²⁰ chlorin e6,Reference Huang, Xu, Lin, Wang, Wang, Zhang, Zhou, Guo and Cui⁴⁰ and camptothecin.Reference Zhang, Xia, Zhao, Liu and Zhang¹¹ The adsorption sites for conjugated molecules are believed to be unfunctionalized sp ²graphenic domains that exist as a patchwork between oxygenated sites on the GO basal plane.

DNA/RNA interactions

The most active area of research on GFN biomolecular interactions involves those with DNA and RNA.Reference Wang, Li, Hu, Lin, Li and Lin^21, Reference Bao, Pan, Ping, Sahoo, Wu, Li, Li and Gan^41, Reference Ren, Wang, Zhang, Zhou, Xu, Zheng and Guo^43, Reference Xu, Wu, Sun, Bai and Shi^45, Reference Lu, Zhu, Li, Liu, Chen and Yang⁴⁹ GFNs show several unique modes of interaction with DNA and RNA, including intercalation into DNA by GFNs of small lateral dimension (Figure 2c),Reference Ren, Wang, Zhang, Zhou, Xu, Zheng and Guo⁴³ preferential adsorption of single-stranded (ss) over double-stranded (ds) forms (Figure 2d),Reference Wu, Kempaiah, Huang, Maheshwari and Liu^44, Reference Lu, Zhu, Li, Liu, Chen and Yang⁴⁹ and steric protection of adsorbed nucleotides from attack by nuclease enzymes.Reference Lu, Zhu, Li, Liu, Chen and Yang⁴⁹ Ren et al.Reference Ren, Wang, Zhang, Zhou, Xu, Zheng and Guo⁴³ reported DNA cleavage under conditions where Cu²⁺ is present but is not active on its own. It appears that this behavior relies on a combination of the planar single-atom-thick GO structure, which can be inserted in the molecular spaces between base pairs, and the peripheral COOH groups that provide sites for Cu²⁺ binding (Figure 2c).

Selective adsorption of ssDNA over dsDNA and protection from degradative enzymes make GO attractive in DNA or RNA delivery and sensing, especially when used in the form of <100-nm “nanosheets” that have the advantages of good dispersibility, colloidal stability, cell uptake, and low toxicity. The preferential binding of ssDNA to GO is believed to reflect the role of GO–base interactions in ssDNA adsorption. DNA is a polyanion and is electrostatically repelled from GO, but the DNA bases can bind to graphenic surfaces (Figure 2d) through hydrophobic forces and π–π stacking.Reference Wu, Kempaiah, Huang, Maheshwari and Liu⁴⁴ These latter attractive forces can overcome the electrostatic repulsion, especially at high ionic strength, where electrolytes shield the charges, or at low pH, where the charge on GO is reduced by protonation. In dsDNA, the bases are protected inside the double helix (Figure 2d), and the outer charged phosphate groups show low affinity for GO surfaces.

Catalysis of oxidative reactions

Oxidative stress is a common toxicity pathway for nanomaterials and has been reported to occur upon exposure of A549 lung-cell cultures to GO.Reference Chang, Yang, Liu, Dong, Wang, Cao, Liu and Wang⁵⁰ Nanosurfaces can generate ROS, which are intermediates along the reduction pathway of molecular oxygen to water, and include HO•, O₂^•–, and H₂O₂. Recently, Liu et al.Reference Liu, Sen, Liu, Kulaots, Geohegan, Kane, Puretzky, Rouleau, More, Palmore and Hurt⁴⁶ reported that a range of carbon nanomaterials, including GO, deactivated the antioxidant glutathione by catalyzing its reaction with O₂. The primary ROS in this study are bound oxygen complexes on edge or defect sites (Figure 2e), which oxidize the biological target, glutathione. Graphene oxide has significant catalytic activity for this model reaction and could thus mediate oxidative damage in living systems, although it shares this property with other members of the carbon nanomaterial family such as nanotubes and carbon black.

Occupational exposures

Some graphene materials are similar in size and shape to known platelike minerals such as talc, mica, slate, and Fuller’s earth,Reference Churg and Green⁵³ although their chemistry is very different. Mining and manufacturing using these naturally occurring minerals can induce scattered areas of dust accumulation and lung granulomas following inhalation into the respiratory tract.Reference Churg and Green⁵³ The conducting airways of the lungs and air spaces or alveoli are covered by macrophages, which are the initial defense (after physical barriers) against inhaled particulates and microorganisms.Reference Geiser⁵⁴ As illustrated in Figure 4, macrophages rapidly engulf or phagocytize platelike GFNs of small lateral dimension.

Figure 4.

Macrophage interactions with carbon nanomaterials. (a) Partial uptake of an FLG sheet 20 layers thick and 5 μm in lateral dimension. (Scanning electron micrograph, 10,500× magnification.) (b) Complete uptake of FLG sheets, 20–30 layers thick and 550 nm in lateral dimension. (c) Attachment of macrophages to the surfaces of a single FLG sheet, 10 layers thick and 25 μm in lateral dimension. Parts (b) and (c) reproduced with permission from Reference Reference Sanchez, Jachak, Hurt and Kane1. ©2012, American Chemical Society. (d) Activated macrophages in nonadherent, three-dimensional cultures aggregate around carbon nanotubes to form a granuloma. Images (b)–(d) are 0.5-μm plastic-embedded sections, toluidine blue stain, light microscopy, 1000× magnification.Reference Allen, Kichambare, Gou, Vlasova, Kapralov, Konduru, Kagan and Star^55, Reference Compton and Nguyen⁵⁶ Part (d) reproduced with permission from Reference Reference Sanchez, Weston, Yan, Hurt and Kane14. ©2011, BioMed Central Ltd.

Following phagocytosis, smaller particulates are sequestered in acidic (pH ∼4.5) cytoplasmic vesicles called lysosomes that contain hydrolytic enzymes capable of degrading biological macromolecules including lipids, proteins, carbohydrates, and nucleic acids.Reference Haas⁵⁷ If inhaled microorganisms and particulates cannot be killed or degraded in lysosomes, additional macrophages are recruited from the peripheral blood and undergo activation.Reference Lehnert⁵⁸ As illustrated in Figure 4, activated macrophages have prominent surface ruffles and produce a variety of chemical mediators that can amplify the initial inflammatory response.Reference Martinez, Helming and Gordon⁵⁹ Direct delivery of carbon nanotubes into the lungs of rodents induces persistent inflammation and fibrosis or fibrous scarring.Reference Sanchez, Jachak, Hurt and Kane^1, Reference Card, Zeldin, Bonner and Nestmann⁶⁰ It is likely that platelike GFNs that are larger in diameter than a macrophage (∼10 μm) cannot be phagocytized (see Figure 4c) and will induce the macrophages to aggregate into a granuloma and cause sustained inflammation in the lungs.

Recent experimental work has investigated potential lung toxicity of GFNs. Duch et al.Reference Duch, Budinger, Liang, Soberanes, Urich, Chiarella, Campochiaro, Gonzalez, Chandel, Hersam and Mutlu⁶¹ compared the toxicity of graphene flakes and GO using mouse lung macrophages or epithelial cells in vitro. Well-dispersed graphene flakes did not induce toxicity in these lung target cells. In contrast, GO causes excess mitochondrial generation of ROS and cell death by apoptosis. Duch et al. speculated that GO can escape from lysosomes into the cytoplasm and participate in the electron-transport chain located in mitochondria, generating excess ROS and thereby leading to cell death.

Other investigatorsReference Wang, Ruan, Song, Zhang, Wo, Guo and Cui⁶² have also shown that GO is toxic to lung epithelial cells and generates ROS extracellularly even in the absence of cell uptake.Reference Chang, Yang, Liu, Dong, Wang, Cao, Liu and Wang^50, Reference Zhang, Ali, Dervishi, Xu, Li, Casciano and Biris⁶³ Duch et al.Reference Duch, Budinger, Liang, Soberanes, Urich, Chiarella, Campochiaro, Gonzalez, Chandel, Hersam and Mutlu⁶¹ also induced significant acute lung injury and sustained inflammation after a single intratracheal delivery of 50 μg of GO into the lungs of mice. Dispersed graphene did not induce lung injury or inflammation, whereas aggregated graphene blocked the small conducting airways and produced a focal fibrotic scar. FLG nanoplatelets also induce inflammation, incomplete phagocytosis, and aggregation of macrophages following nasopharyngeal aspiration (50 μg) into the lungs or direct injection (5 μg) into the pleural space of mice.Reference Schinwald, Murphy, Jones, Macnee and Donaldson²²

Potential toxicity in biomedical applications

Like other materials based on elemental carbon, GFNs have the potential to be biocompatible, and carbon nanomaterials have been explored for applications as biomedical implants, biological sensors, and drug-delivery devices.Reference Sanchez, Jachak, Hurt and Kane^1, Reference Feng and Liu^15, Reference Liu, Tabakman, Welsher and Dai⁶⁴ In comparison with SWCNTs, GO has a higher specific surface area and can be functionalized, dispersed in biological media, and produced in large quantities at lower cost.Reference Feng and Liu¹⁵ Similarly to other drug-delivery platforms, graphene nanosheets and GO can be coated with polymers such as poly(ethylene glycol) (PEG)Reference Liu, Robinson, Sun and Dai⁴² or chitosanReference Liao, Lin, Macosko and Haynes⁶⁵ to prolong circulation time and enhance biocompatibility. For example, Yang et al.Reference Yang, Zhang, Zhang, Sun, Lee and Liu⁵² produced PEG-coated GO sheets in the size range of 10–50 nm that were nontoxic up to 40 days following intravenous injection in mice. These nanoscale coated GO sheets showed excellent passive uptake into tumor xenografts in mice and allowed successful photothermal tumor ablation.

Nanoscale drug delivery following intravenous administration can lead to systemic distribution to secondary target organs including the liver and spleen, as illustrated in Figure 5. Feng and LiuReference Sanchez, Weston, Yan, Hurt and Kane¹⁴ reported that nanoscale PEG-coated GO sheets initially accumulated in these organs and were then gradually excreted with no indication of toxicity after three months.Reference Yang, Wan, Zhang, Zhang, Lee and Liu⁶⁷ On the other hand, Wang et al.Reference Wang, Ruan, Song, Zhang, Wo, Guo and Cui⁶² and Zhang et al.Reference Zhang, Yin, Peng, Hu, Zhu, Li, Fan and Huang⁶⁸ reported that intravenous injection of uncoated GO sheets induced granulomas in the lungs,Reference Wang, Ruan, Song, Zhang, Wo, Guo and Cui^62, Reference Zhang, Yin, Peng, Hu, Zhu, Li, Fan and Huang⁶⁸ liver, spleen, and kidneys at a dose of 0.4 mg per mouse. Intravenous delivery of GO nanosheets might also induce the breakdown of red blood cells (hemolysis),Reference Liao, Lin, Macosko and Haynes^65, Reference Zhang, Yin, Peng, Hu, Zhu, Li, Fan and Huang⁶⁸ and graphene nanosheets have been found to induce red blood cell aggregation in vitro. Reference Liao, Lin, Macosko and Haynes⁶⁵ These studies confirm the importance of surface functionalization and aggregation state on the potential toxicity of GFNs.

Figure 5.

Routes of exposure, systemic distribution, and elimination of nanomaterials from the body following skin contact, implantation into soft tissues and bone, intravenous injection, inhalation into the respiratory tract, and ingestion. Figure modified from Obderdörster et al.Reference Oberdorster, Oberdorster and Oberdorster⁶⁶

A final consideration regarding the long-term toxicity of GFNs used in biomedical applications is biopersistence. Nonbiodegradable implants with smooth, continuous surfaces induce foreign-body tumors following implantation in rodents.Reference Oppenheimer, Willhite, Danishefsky and Stout⁶⁹ High-surface-area GFNs with smooth surface topography and biopersistenceReference Soldano, Mahmood and Dujardin⁷⁰ have the potential to induce chronic inflammation with persistent generation of ROS that have been linked with the pathogenesis of foreign-body tumors.Reference Okada⁷¹ GFNs can be engineered deliberately to promote biodegradation, as demonstrated for carbon nanotubes.Reference Allen, Kichambare, Gou, Vlasova, Kapralov, Konduru, Kagan and Star^55, Reference Kagan, Konduru, Feng, Allen, Conroy, Volkov, Vlasova, Belikova, Yanamala, Kapralov, Tyurina, Shi, Kisin, Murray, Franks, Stolz, Gou, Klein-Seetharaman, Fadeel, Star and Shvedova^72, Reference Liu, Hurt and Kane⁷³ In addition, GO is susceptible to degradation by enzymatic oxidation.Reference Kotchey, Allen, Vedala, Yanamala, Kapralov, Tyurina, Klein-Seetharaman, Kagan and Star⁷⁴

Other manufacturing issues

The chemical synthesis of GO can lead to explosions associated with Cl₂O gas, which is a byproduct of the use of potassium chlorate as an oxidizer,Reference Marcano, Kosynkin, Berlin, Sinitskii, Sun, Slesarev, Alemany, Lu and Tour⁸⁴ or residual acetone, which is commonly used to clean glassware.Reference Lee, Oh, Ruoff and Park⁸⁵ Like all carbon materials, graphene and GO can be oxidized, but not all forms are easily ignitable. Kim et al.Reference Kim, Cote and Huang³⁷ reported that GO papers containing residual salts from the synthesis process are highly flammable as a result of catalytic oxidation. Local heating can induce GO reduction, the exothermicity of which ignites the paper in a coupled reduction/combustion process. This effect can be suppressed by removing salt using additional purification steps.Reference Kim, Cote and Huang³⁷ As manufacturing volume grows, so will interest in producing graphenes in a cost-effective and sustainable manner. The competing methods (chemical vapor deposition, exfoliation, GO wet synthesis) use very different types of processing and are likely to have very different energy usages, raw material and waste productions, and overall sustainability indices. Safe and sustainable graphene manufacture on a large scale is a promising area for future research and life-cycle assessment.