Developing the next generation of graphene-based platforms for cancer therapeutics: The potential role of reactive oxygen species

Abstract

Graphene has a promising future in applications such as disease diagnosis, cancer therapy, drug/gene delivery, bio-imaging and antibacterial approaches owing to graphene's unique physical, chemical and mechanical properties alongside minimal toxicity to normal cells, and photo-stability. However, these unique features and bioavailability of graphene are fraught with uncertainties and concerns for environmental and occupational exposure. Changes in the physicochemical properties of graphene affect biological responses including reactive oxygen species (ROS) production. Lower production of ROS by currently available theranostic agents, e.g. magnetic nanoparticles, carbon nanotubes, gold nanostructures or polymeric nanoparticles, restricts their clinical application in cancer therapy. Oxidative stress induced by graphene accumulated in living organs is due to acellular factors which may affect physiological interactions between graphene and target tissues and cells. Acellular factors include particle size, shape, surface charge, surface containing functional groups, and light activation. Cellular responses such as mitochondrial respiration, graphene-cell interactions and pH of the medium are also determinants of ROS production. The mechanisms of ROS production by graphene and the role of ROS for cancer treatment, are poorly understood. The aim of this review is to set the theoretical basis for further research in developing graphene-based theranostic platforms.

Keywords: Bioimaging; Graphene; Photodynamic therapy; Reactive oxygen species; Singlet oxygen; Theranostics.

Fig. 1

Schematic representation of a typical theranostic platform for the combined use of a range of imaging and therapeutic approaches. Imaging modalities include: ultrasonography, positron electron tomography, fluorescence imaging, magnetic resonance imaging and single-photon emission computed tomography. Therapeutic approaches include: drug delivery, photothermal therapy, photodynamic therapy, or a combination of two therapies. Based on its unique properties, graphene can be employed as a theranostic agent that combines the capabilities of diverse imaging and therapeutic modalities to target tumors.

Fig. 2

Summary of structural models of various derivatives of graphene. (a) Graphene, (b) graphene oxide (GO), (c) reduced graphene oxide, (d) porous graphene, (e) graphene quantum dots and (f) three dimensional graphene foam. Graphene is a sp² hybridized model of carbon atoms in a repeated manner, forming a regular lattice structure (as shown in panel a), while GO and reduced GO have functional groups and defects in their basal planes (panels b and c). The physicochemical properties and structures of different graphene variants depend on the fabrication method and conditions. The presence of both defects and functional groups provides potential advantages for the efficient utilization of graphene variants in the production of ROS. The chemical exfoliation method is thought to be an efficient route for synthesizing graphene on a large scale and at low cost. Porous graphene is a graphene sheet that is missing carbon atoms from its plane. The various forms of porous graphene provide fascinating materials for biological applications owing to their high specific surface areas, hydrophobic nature and biocompatibility. Graphene nanopores usually have pore sizes of 1–30 nm. Pores and vacancies can clearly be seen in the porous graphene sheet, as represented in panel (d). Graphene quantum dots are luminescent nanocrystals having a size less than 50 nm. These have attractive properties and potential applications in cancer diagnosis and treatment. Water soluble graphene quantum dots, shown in panel (e), have functional groups (C–OH, C=O, C–O–C, C–H) on their surface. Three-dimensional graphene networks in the form of a foam, sponge or aerogel have recently been assembled from individual graphene sheets using chemical vapour deposition templated methods, which also preserve the unique properties of individual graphene sheets. [Panel (f) is adapted from , with permission of MDPI Publishing Group, Copyright 2015].

Fig. 3

Schematic illustration of the potential mechanisms by which reactive oxygen species (ROS) are associated with the cellular toxicity of graphene. Graphene may affect biological behavior at the cellular, subcellular, protein and gene levels. The toxicity of graphene depends on its physicochemical interactions and its accumulation in specific organs. Uptake of graphene into specific organs also affects cell function as a result of cellular changes within the organs. The deposition, distribution and clearance of graphene after entering into a living system is a major knowledge gap in understanding the toxicity of graphene. Graphene circulating in the bloodstream is internalized into cells through the plasma membrane. The plasma membrane is a selectively permeable membrane that transfers materials such as ions and nano-sized proteins. Graphene (depending on its size, shape, and surface chemistry) enters the cell via different pathways such as clathrin/caveolar-mediated endocytosis, phagocytosis, macropinocytosis, and pinocytosis and exits the cell via the pathways of lysosome secretion, vesicle-related secretion, and non-vesicle-related secretion. The nature of plasma membrane interaction with graphene determines the fate of graphene in a wide range of potential applications with high biocompatibility, including drug- and gene-delivery, photothermal and photodynamic therapy. This interaction may lead to the possibility of events such as adsorption or incorporation of graphene onto the surfaces of cells. Furthermore, the entrapped biomolecules on the surface of graphene, when graphene is present within the extracellular matrix, may influence the tertiary structure of a protein - resulting in the formation of a protein-graphene interface and malfunction. The extracellular mechanisms causing the accumulation of graphene in the extracellular matrix and the subsequent effects of graphene on the extracellular matrix remain undefined. Graphene-induced ROS may cause oxidative stress, loss of cell function, mitochondrial damage, initiation of lipid peroxidation, covalent chemical modifications of nucleic acids, DNA-strand breaks, induction of gene expression via the activation of transcription factors, and modulation of inflammation via signal transduction - leading to toxicity, cell death and genotoxicity. The specific minerals in the secondary antioxidants are being referred to selenium, zinc, molybdenum, iron and copper. The antioxidant defence system is overwhelmed by high levels of ROS, leading to oxidative stress, inflammation and toxicity. One potential way to minimize the toxicity of graphene is to functionalize the graphene with biodegradable agents.

Fig. 4

Cell signaling and molecular targets of ROS in cancer. ROS may induce both transcriptional factors/activators and genes associated with tumor suppression: HIF-1α (hypoxia-inducible factor-1 alpha); NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells); PTEN (phosphatase and tensin homolog deleted on chromosome 10); AP-1 (activator protein-1); Hh (hedgehog protein); STAT3 (signal transducer and activator of transcription 3); Rb (retinoblastoma protein); Nrf2 (nuclear factor (erythroid-derived 2)-like 2); Sp1 (specificity protein 1). NF-κB and AP-1 are transcription factors that play key roles in the expression of many genes involved in inflammation as well as many other significant events such as embryonic development, lymphoid differentiation and apoptosis. HIF-1α plays an essential role in embryonic vascularization and tumor angiogenesis. Nrf2, a redox-sensitive transcription factor, regulates genes which bind antioxidant response elements in DNA. PTEN is a tumor suppressor gene, which is deleted or mutated at high frequency in a large number of cancers. Rb protein is a tumor suppressor gene which controls cell cycle progression. Sp1 is a transcription factor which contributes to overexpression of MDM2 (mouse double minute 2 homolog) in rhabdomyosarcoma tumors. Stat 3 is a transcription factor which plays an important role in cell growth and apoptosis. ROS-mediated signaling through activation of these transcription factors controls the expression of genes involved in inflammation, metastasis, cell proliferation and tumor angiogenesis, as well as tumor cell death or survival.

Fig. 5

Schematic representations of the mechanism involved in singlet oxygen production leading to programmed cell death induced by combined photodynamic and photothermal therapies using a graphene nanocomposite photosensitizer. Photodynamic therapy (PDT) refers to the use of a non-toxic compound called a photosensitizer and a special laser light to kill cancer cells, while photothermal therapy (PTT) uses the heat generated from the absorbed optical energy by light-absorbing nanoparticles embedded within tumors to ablate tumor cells. Panel (a) shows a schematic illustration of the mechanisms of singlet oxygen (¹O₂) generation by a photosensitizer, in the form of a Jablonski diagram representing the electronic states of a photosensitizer after light absorption, followed by energy transfer to an oxygen molecule to generate ¹O₂. The photosensitizer displays intersystem crossing to the triplet state when the photosensitizer is excited to the singlet state. The electronic states are shown in the diagram. Internal conversion: transitions between states of similar electronic spin, where the electronic states are singlet and triplet. Fluorescence: the emitted photon has energy resembling the energy difference between the initial and final states of the photosensitizer. The emitting and final states have similar electronic spin states, either singlet or triplet. Intersystem crossing: the change of electronic spin in the excited state, from singlet to triplet. Phosphorescence: the emitted photon has energy resembling the energy difference between the initial and final states of the photosensitizer. The emitting and final states have different electronic spin states, such as one in the singlet state and the other in the triplet state. Panel (b) is a schematic illustration of the mechanism of cancer cell killing induced by a functionalized hybrid of folic acid (FA), polyethylene glycol (PEG) and C₆₀ (a spherical fullerene molecule with the formula C₆₀ called buckminsterfullerene) non-covalently conjugated to GO for synergistic combined photothermal therapy and photodynamic therapy. Thus, the functionalized hybrid consists of FA-GO-PEG/C₆₀ [FA (cancer targeting moiety) and C₆₀ (photosensitizer) conjugated to PEGylated graphene oxide]. Functionalized GO was exposed to light sources with wavelengths of 532 and 808 nm for enhanced cellular uptake of C₆₀ in cancer cells. The GO nanocomposite showed effective cell apoptosis and death and exhibited a synergistic effect of combined photodynamic and photothermal therapies. [Panel (b) is adapted from , with permission of the Royal Society of Chemistry, Inc., Copyright 2015].