Graphene: Properties, Biomedical Applications and Toxicity

TANDABANY C. DINADAYALANE, DANUTA LESZCZYNSKA AND JERZY LESZCZYNSKI

1.1 Introduction

Among numerous commercial endeavors, nanotechnology is regarded as the key technology of the 21st century. It provides novel products and facilitates applications of innovative techniques in medicine, pharmacy, computer technology, and sensing. Therefore, it holds promise for potential global socioeconomic benefits. In 2011, there were about 1100 commercial products that include nanomaterials. It is a common belief that nanotechnology could assist in solving many global problems that society faces. These include environmental and health concerns of the fast-growing human population, as well as access to clean water and affordable energy. The quickly growing applications of nanomaterials are due to their unique properties which offer advantages over conventional materials.

The variety of various nanomaterials is too big to cover in a single chapter. Therefore, we decided to focus on one particular class of species and discuss in details its characteristics and applications. Since the 2010 Nobel awards validate the importance of graphene not only in basic research but also in various commercial applications, we selected this nanomaterial as the main subject of our chapter.

The demand for carbon nanostructures, particularly carbon nanotubes (CNTs) and graphene, is increasing rapidly in electrical, mechanical, and biomedical applications. This is due to their outstanding thermal, electrical, mechanical, optical and other unique properties. Although the intense interest and continuing experimental success of graphene-based devices facilitate their various applications, the reliable production of high quality samples of graphene on a large scale is very difficult. At present, great efforts have been made toward the preparation of graphene nanosheets. Among them, the chemical reduction of exfoliated graphene oxide (EGO) is the most commonly used approach due to its low cost for large-scale production. In the case of carbon nanotubes, closely related to graphene, controlling their size and diameter is still very challenging. The availability of carbon nanotubes, both in quality and quantity, has stimulated the worldwide pursuit of carbon nanotubes for technological applications. Nevertheless, carbon nanotubes (especially single-walled carbon nanotubes (SWCNTs)) are still quite expensive. To assist experimental studies, the structures, reactivities, and functionalization of defect-free and Stone–Wales defective SWCNTs have been investigated by our group, using quantum chemical calculations.

The morphology of graphene is different from that of CNTs; for example, the length of CNTs influences their toxicity but graphene and graphene oxide (GO) do not have a "length". An important similarity between these carbon nanomaterials is that both graphene/graphene oxide and carbon nanotube structures vary according to the synthetic processes employed. Such processes can also change their physical properties, including dispersity, surface functionality, and their toxicity. In the materials science world, carbon nanostructures such as fullerenes, carbon nanotubes and graphene are famous for their small dimension and unique architecture, and several possible applications in diversified areas. In addition to these carbon nanostructures, scientists now produce a plethora of carbon-based nanoforms such as 'bamboo' tubes, 'herringbone' and 'bell' structures. Figure 1.1 shows a "family tree" of carbon nanoforms that were obtained by applying various transformations to graphene (details of operations are provided in the figure). Though the diagram is non-exhaustive (primarily for clarity), this chart is useful to classify the nanoforms by morphology and provides a first step towards a standardized nomenclature.

The research involving graphene has grown at a spectacular pace in the last few years. Several potential applications have been proposed for graphene. These include conductive and high-strength composites, energy storage and energy conversion devices, sensors, field emission displays and radiation sources, hydrogen storage media, and nanometre-sized semiconductor devices, probes, and interconnect (Scheme 1.1). Impressive advances have been made in realizing some of the applications of carbon nanotubes and graphene. Graphene is a possible replacement material in applications where carbon nanotubes are presently used. A recent study has revealed that graphene-based liquid crystal devices (LCDs) showed an excellent performance with a high contrast ratio. Thus, LCDs might be the first realistic commercial application of graphene. Like carbon nanotubes, the unique properties of graphene offer a wide range of opportunities and application potential for biology and medicine (Scheme 1.1). Bioapplications of carbon nanotubes and graphene have attracted much attention recently.

Significant recent development and progress in the use of graphene-based materials for biosensors, drug and other delivery systems, and bioimaging have generated much excitement in the research community. In the last few years, considerable attention has been paid to the health risks of carbon nanomaterials due to rising global production and the wide range of proposed applications.

The lungs and skin are regarded as the two main potential exposure sites during the manufacture and handling of carbon nanotubes. The high ratio between the length and diameter of carbon nanotubes and their low solubility in aqueous media make them potentially biopersistent and may lead to toxic effects similar to those seen with asbestos. Carbon nanotubes might induce lung cancer and mesothelioma in a similar manner to that of asbestos. Hence, an understanding of the occupational health, public safety and environmental implications of carbon nanomaterials is required. Some recent studies have reported the in vivo biodistribution and toxicity of carbon nanotubes and graphene. In this chapter, we briefly present various aspects related to the toxicity of graphene-based nanomaterials.

1.2 Structure and Properties of Graphene

Carbon has a unique feature of forming a chemically stable two-dimensional (2D), one-atom thick sheet called graphene. Each carbon atom in graphene is covalently bonded to three other carbon atoms with sp2 hybridization. Graphene is the thinnest known material and the strongest material ever to be measured. It can sustain current densities that are six orders of magnitude higher than those of copper. Furthermore, it has very high thermal conductivity and stiffness, and is impermeable to gases. Interestingly, the honeycomb network of graphene is also the basic building block of other important carbon nanostructures. Hence, graphene may be considered as the mother of graphite, fullerene and carbon nanotubes (Scheme 1.2). Graphene can also be considered as the final member of the series of fused polycyclic aromatic hydrocarbons, such as naphthalene, anthracene and coronene.

For several years, scientists thought that planar graphene could not exist in a free state since it is unstable compared to curved structures such as soot, nanotubes and fullerenes. Surprisingly, in 2004, Novoselov et al. prepared graphitic sheets including a single graphene layer and studied their electronic properties. Just a few years later, in 2010, Andre Geim and Konstantin Novoselov were awarded the Nobel Prize for their groundbreaking experimental isolation of single-layer graphene. The experimental realization of graphene generated vast interest in the areas of fundamental physics, materials science and engineering. Researchers have been looking for possible applications of graphene in high-speed and radio-frequency logic devices, thermally and electrically conductive reinforced composites, sensors, transparent electrodes for displays and solar cells, and biomedicine. Numerous graphene-based biosensing devices and techniques based on various mechanisms have been developed in the past few years. Graphene is obviously a prospective material for nanoelectronics. The electron transport in graphene was described by a Dirac-like equation. The studies pertinent to the chemistry of graphene sheets have also been reported. The growth and isolation of graphene have been recently reviewed. Research in the area of graphene material has been rapidly growing due to the recent advances in technology for the growth, isolation and characterization of two-dimensional carbon sheets.

Graphene sheets need not always be perfect. Various defects such as Stone–Wales (SW), vacancies, and pore defects can occur in the graphene sheet. Like the creation of vacancies by knocking atoms out of the graphene sheet, surplus atoms can be found as ad-atoms on the graphene surface. An ad-dimer or inverse Stone–Wales (ISW) defect is characterized by two adjacent five-membered rings instead of two adjacent seven-membered rings in a Stone–Wales defect. Experimental observations of defects in graphene were reported.

Zettl and co-workers showed the direct image of Stone–Wales defects in graphene sheets using high resolution transmission electron microscopy (HRTEM) and explored their real-time dynamics. They found that the dynamics of defects in extended, two-dimensional graphene membranes are different from in closed-shell graphenes such as nanotubes or fullerenes. The results of our computational study (calculations at the B3LYP/6-31G(d) level) indicate that the Stone–Wales defective graphene sheet is 83.3 kcal mol-1 less stable than the defect-free graphene sheet (Scheme 1.3). Figure 1.2 depicts some of the common defects and HRTEM images of defects in a graphene sheet. The characteristics of typical defects and their concentrations in graphene sheets are unclear.

1.2.1 Biomedical Applications of Graphene

Recently, graphene has showed promise similar to carbon nanotubes in various biomedical applications such as drug delivery and cancer therapy. Graphene can provide a larger specific surface area than other commonly used carbon nanomaterials and forms strong π–π interactions with the drug molecules, which can therefore act as a good candidate for drug loading. Very recently, in vivo cancer treatment using graphene has been realized in animal experiments. Functionalized graphene sheets have been mostly used for biomedical applications. PEGylated (PEG – polyethylene glycol) nanoscale graphene oxide (NGO-PEG), which has a high stability in physiological solutions, can be utilized for effective loading of aromatic anticancer drugs such as doxorubicin (DOX) and water-insoluble SN38. It is accomplished via π–π stacking (Figure 1.3a). The unique 2D shape and ultra-small size (down to 5 nm) of NGO-PEG may offer interesting in vitro and in vivo behaviors.

The aromatic molecule SN38 is a camptothecin (CPT) analogue and a potent topoisomerase I inhibitor. The free SN38 was mentioned to be insoluble in water, but the NGO-PEG–SN38 complex exhibited excellent aqueous solubility. The experimental study revealed no loading of SN38 on the PEG polymer in a solution free of NGO. Furthermore, the hydrophobic and π–π interactions (between SN38 and aromatic regions of the graphene oxide sheet) were attributed to the binding of SN38 onto NGO-PEG. The water-soluble NGO-PEG–SN38 exhibited high potency with IC50 values of about 6 nM for HCT-116 cells, which is ~1000-fold more potent than CPT-11 (an FDA-approved SN38 prodrug for colon cancer treatment). Hence, NGO-PEG–SN38 has a high potential for killing cancer cells in vitro with a human colon cancer cell line HCT-116. The potency of NGO-PEG–SN38 was reported to be similar to that of free SN38 dissolved in DMSO. Liu et al. have also observed the high potency of NGO-PEG–SN38 with various other cancer cell lines in their tests.

The extremely large surface area of graphene, with every atom exposed on its surface, allowed for ultra-high drug loading efficiency on NGO-PEG. The terminals of PEG chains were available for the conjugation of targeting ligands such as antibodies. This facilitated targeted drug delivery to specific types of cancer cell (Figure 1.3b). Rituxan (CD20+ antibody) conjugated NGO-PEG was used to target specific cancer cells for selective cell killing. The drug delivery research revealed that approximately 40% of DOX loaded on NGO- PEG was released over 1 day in an acidic solution of pH 5.5. This phenomenon was attributed to the increased hydrophilicity and solubility of DOX at this pH. Compared to single-walled carbon nanotubes for drug loading via π–π stacking, NGO is advantageous in terms of its low cost and ready scalability. Yang et al. have studied the in vivo behaviors of nanographene sheets (NGS) with polyethylene glycol (PEG) coating by a fluorescent labeling method. They have labeled NGS-PEG with Cy7, which is a commonly used near- infrared fluorescent dye, in order to study the in vivo behaviors of NGS. Interestingly, the majority of Cy7 dye was covalently conjugated to NGS-PEG via the formation of an amide bond instead of p-stacking physisorption. BALB/c mice bearing 4T1 murine breast cancer tumors, nude mice bearing KB human epidermoid carcinoma tumors, and U87MG human glioblastoma tumors were intravenously injected with NGS-PEG–Cy7. Yang et al. have observed a prominent uptake of NGS in the tumor with relatively low quantity in other parts of the mouse body after 24 h post-injection for all three types of tumor models.

The graphical and pictorial illustrations of in vivo photothermal therapy study using intravenously injected NGS-PEG are given in Figure 1.4. Tumor growth curves of different groups after treatment were given. The tumor volumes were normalized to their initial sizes. There were six mice in the untreated group, ten mice in the 'laser only' group, seven mice in the 'NGS-PEG only', and ten mice in the 'NGS-PEG + laser' groups. While injection of NGS-PEG by itself or laser irradiation on uninjected mice did not affect tumor growth, tumors in the treated group were completely eliminated after NGS-PEG injection and followed by near-infrared (NIR) laser irradiation.

The laser-irradiated tumor on NGS-PEG injected mouse was completely destroyed. The in vivo study revealed that the high kidney uptake of NGS-PEG–Cy7 might indicate possible renal excretion of NGS with small sizes (Cy7 fluorescence was indeed detected in the mouse urine) but this needs further validation. Nude mice bearing KB or U87MG tumors also showed high tumor and kidney uptake of NGS (see Figure 1.4d). Mice in the three control groups showed average life spans of ~16 days, while mice in the treated group were tumor-free after treatment (NGS injection + NIR laser irradiation) and survived over 40 days without a single death (Figure 1.4b). This finding demonstrates the excellent efficacy of NGS-based in vivo photothermal therapy.