Biomedical Applications of Graphene

Biomedical Applications of Graphene

9 Biomedical Applications of Graphene Jidong Shi, Ying Fang CHAPTER OUTLINE 9.1 Graphene-Based Biosensors ...

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9 Biomedical Applications of Graphene Jidong Shi, Ying Fang CHAPTER OUTLINE 9.1 Graphene-Based Biosensors ....................................................................................................... 216 9.1.1 Graphene-Based Electrophysiological Sensors ................................................................ 216 9.1.2 Graphene-Based Affinity Sensors..................................................................................... 220 9.1.3 Graphene Nanopores for the Detection of DNA............................................................ 220 9.1.4 Graphene-Based Tissue Engineering................................................................................ 223 9.2 Graphene Derivative-Based Functional Carriers ...................................................................... 224 9.2.1 Graphene Oxide-Based Fluorescent Biosensing.............................................................. 224 9.2.2 Graphene Oxide-Based Bioimaging ................................................................................. 226 9.2.3 Graphene Oxide-Based Nanocarrier for Drug Delivery.................................................. 227 9.3 Biosafety of Graphene ................................................................................................................ 228 9.4 Conclusions and Outlooks .......................................................................................................... 229 References........................................................................................................................................... 229

Graphene is a two-dimensional (2D) nanomaterial composed of sp2 hybridized carbon atoms and has attracted considerable attention due to its unique physical and chemical properties. The biomedical applications of graphene and its derivatives have been developing rapidly in recent years. For example, graphene-based field-effect transistors (FETs) have been applied for the sensitive detection of both biomolecules and electrophysiological signals. In most of these studies, graphene sheets, with high-quality crystal structures and excellent electrical and mechanical properties, were produced either through mechanical exfoliation or chemical vapor deposition (CVD). On the other hand, graphene derivatives, such as graphene oxide (GO), have been widely applied in biological imaging, drug delivery, and cancer therapy, due to their large surface area and ease of preparation and functionalization. GO nanosheets, produced by vigorous oxidation of graphite, are usually 13 layers with lateral sizes ranging from a few nanometers to several hundred nanometers. GO is photoluminescent due to the presence of isolated sp2 domains and edges/defects. GO nanosheets can be readily modified through either ππ stacking interactions or their abundant oxygencontaining groups to facilitate targeted imaging and drug delivery. By the reduction of GO to reduced GO (rGO), the oxygen-containing groups on the nanosheets are partly removed, Graphene. DOI: http://dx.doi.org/10.1016/B978-0-12-812651-6.00009-4 Copyright © 2018 Tsinghua University Press Limited. Published by Elsevier Inc. All rights reserved.

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which leads to improved electrical and thermal conductivity of rGO. Previous studies have used rGO as building blocks to fabricate transistor-based biosensors. Nevertheless, rGO still contains a large amount of lattice defects and significantly differs from pristine graphene. In this chapter, we will introduce the biomedical applications of graphene and its derivatives. Section 9.1 discusses recent advances in biosensors based on graphene FETs. Section 9.2 describes functional carriers based on graphene derivatives for biological imaging and drug delivery. In view of the growing interests in the biomedical applications of graphene and its derivatives, their biosafety will be discussed in Section 9.3.

9.1 Graphene-Based Biosensors Single-crystalline graphene with hexagonal carbon lattice has a Dirac cone in the band structure and exhibits unprecedented electronic properties [1]. In addition, the theoretical fracture strain of single-crystalline graphene is as high as 13% [2]. Combined with its single atomic layer thickness, graphene has been identified as a prominent candidate in flexible electronics. High-quality graphene can be prepared through mechanical exfoliation [3], chemical vapor deposition (CVD) [4], or epitaxial growth on Si-terminated 6H-silicon carbide (SiC) wafers [5]. The carrier mobility of mechanically exfoliated graphene can reach as high as 106 cm2/V/s, which is essential for high sensitivity detection of biomolecules and electrophysiological signals. However, the size of the exfoliated graphene sheets is usually limited to hundreds of micrometers [6]. In comparison, graphene grown by CVD or epitaxial methods has both relatively high carrier mobility ( . 1000 cm2/V/s) and large areas [7], which facilitates process scalability and the fabrication of graphene-based biosensor arrays.

9.1.1 Graphene-Based Electrophysiological Sensors Sensitive recording of electrophysiological signals is central to understanding the bioelectrical activities of the body, such as in the brain, heart, and muscles. The lipid bilayer membranes of the neurons and cardiomyocytes consist of a number of ion channel proteins which regulate the movement of selected ions across the cellular membranes. During an action potential, ions flow across the neuronal or cardiomyocyte membrane, which leads to both intracellular and extracellular potential spikes. In the brain, spike waves propagating among neural circuits carry out the diverse functions of the brain, including learning, memory, and cognition. The spike waves propagating through the heart control the dynamics of cardiac contraction/relaxation and the transportation of nutrients and oxygen. Metal electrode arrays (MEAs) have been widely applied for multisite and long-term recording of neuronal or cardiac electrophysiological signals [8]. The extracellular potential signals can induce an electric current through the depolarization of the metal surface. However, the development of MEAs encounters several technical bottlenecks. Firstly, the mechanical mismatch between the planar metal electrode and the cell leads to a low electrical coupling coefficient of approximately 0.1%, which results in poor signal-to-noise ratio (SNR). In addition, the spatial resolution of MEAs is limited to tens of micrometers. The reduction of the sizes of the metal electrodes can increase their spatial resolution. However,

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size reduction leads to increased impedance at the interface between the electrode and the cell, which thus results in increased thermal noise and decreased SNR of MEAs [9]. As an alternative to MEAs, electrolyte-oxide silicon field-effect transistors (EOS-FETs) have been developed for multisite recording of electrophysiological signals. The extracellular potential spikes can change the carrier density in semiconducting silicon through the field effect, which leads to an electric signal in the transistor (Fig. 9-1A) [9]. In particular, FETs based on semiconductor nanomaterials offer the potential for sensitive detection of electrophysiological signals at high spatial resolution. For example, Lieber et al. pioneered in using silicon nanowire FETs to record electrophysiological signals from both neurons [11] and cardiomyocytes [12]. On the other hand, Rogers et al. fabricated flexible transistor arrays by using single-crystalline silicon nanoribbons formed by chemical etching and transfer printing, in which both neuronal and epicardial wave mapping have been successfully (A)

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FIGURE 9-1 Graphene-based electrophysiological sensors [10]. (A) Circuitry model of graphene transistorcell interface. (B) Schematic of the tight contact between a suspended graphene transistor and a cell. (C) Recorded current signals from a heart by a graphene transducer, with an active area of 17.5 µm2, before (black) and after suspension (red, gray in print versions), respectively. The right panels are the magnified view of single spikes.

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demonstrated [13,14]. However, silicon nanomaterials suffer from low chemical stability under physiological conditions, which leads to transistor deterioration and failure during long-term recording. In comparison, graphene offers excellent chemical stability under physiological conditions. The high surface area and extraordinary carrier mobility of graphene are advantageous to achieve high sensitivity in electrophysiological recording. In addition, the high transparency and excellent mechanical properties make graphene an attractive candidate for transparent and flexible electronics. In 2010, Fang and Lieber et al. showed that electrophysiological sensors based on exfoliated single-layer graphene sheets could form robust bioelectronic interfaces with cultured cardiomyocytes [15]. The extracellular potential signals of the cardiomyocytes served as the gate input, which modulated the carrier concentrations of graphene and produced a signal in the drain current of the graphene transistor. The recorded current spikes were synchronized with the spontaneous beating of cardiomyocytes with SNR . 4. Furthermore, the polarity of the current signal was flipped when graphene was tuned from p-type to n-type characteristics, owing to the unique bipolar characteristic of graphene. Graphene FETs are usually fabricated on silicon oxide substrates. Former studies have shown that the charge traps and remote optical phonons in the oxide layer strongly scatter the charge carriers of graphene, which results in large 1/f noise and seriously degrades the electrical performance of graphene FETs. To improve the SNR of graphene sensors, Fang et al. fabricated suspended graphene FETs in aqueous solution through an in situ etching technique [16]. The results showed that the SNR of the graphene sensors was greatly improved as a result of concomitantly increased mobility and decreased 1/f noise in graphene by suspension in solution. In 2013, Fang et al. systematically investigated the dependence of sensitivity on graphene size under physiological conditions. They showed that the sensitivity of suspended graphene FETs follows a square root scaling on the area of graphene. This scaling rule can be understood in terms of the disorder potentials induced by the thermal fluctuations of water molecules. In addition, they showed that suspended graphene FETs can form tight bioelectronic interfaces with cells (Fig. 9-1B). As a result, suspended graphene FETs demonstrated greatly improved SNR during electrophysiological recording (Fig. 9-1C) [10]. Large-area graphene prepared by CVD methods offers the advantages of low cost and scalability. In 2011, Garrido et al. fabricated graphene transistor arrays using CVD-grown graphene films (Fig. 9-2A) [17]. Each array consisted of 16 graphene strips with lateral dimension of 20 µm 3 10 µm (Fig. 9-2B). Cardiomyocyte-like HL-1 cells were then cultured on the graphene transistor array. Parallel measurement of the cellular bioelectrical signals was demonstrated with performance comparable to the state-of-the-art MEAs (Fig. 9-2C). Recently, Fang et al. developed a flexible, highly crumpled all-carbon transistor array for brain activity recording. The all-carbon transistors were chemically synthesized by the seamless integration of graphene channels and hybrid graphene/carbon nanotube (CNT) electrodes (Fig. 9-3A) [19]. Owing to the perfectly matched mechanical properties between graphene and CNTs, the all-carbon transistors maintained their structural integrity and stable electronic properties under large compression deformation (Fig. 9-3B). As a result, highly crumpled all-carbon transistors with substantially compressed projected area and

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large active graphene area were achieved, permitting a sixfold increase in spatial resolution. The highly crumpled all-carbon transistors provided both high sensitivity and improved spatial resolution during brain activity recording (Fig. 9-3C,D) [18].

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9.1.2 Graphene-Based Affinity Sensors Affinity biosensors based on FETs allow label-free and real-time detection of biological molecules, which has attracted numerous attentions in areas such as clinical diagnosis and drug discovery. A graphene-based affinity biosensor uses a surface-immobilized receptor such as enzyme, antibody, or single-stranded nucleic acid to selectively capture a target biomolecule from solution. This recognition process is then converted into an electrical signal by the graphene transistor, which thus allows quantitative or semiquantitative analysis of the target biomolecule. The surface of the graphene is usually functionalized with the receptor molecules through ππ stacking. This noncovalent functionalization method does not disrupt the crystalline structure or degrade the electrical properties of graphene, which is essential to maintain the high sensitivity of the sensors. Graphene-based affinity biosensors have been developed for the detection of proteins based on either the specific aptamerprotein reaction or antibodyantigen reaction. For example, Matsumoto et al. immobilized Immunoglobulin E (IgE) aptamers on the surface of graphene FETs for label-free detection of the IgE protein [20]. 50 -amino terminated anti-IgEaptamer was immobilized on the graphene surface by using 1-pyrenebutanoic acid succinimidyl ester as a linker. The pyrenyl group of the linker absorbs strongly on the basal plane of graphene via ππ stacking, and the succinimidyl ester group of the linker reacts with the amine group of the aptamer to form a strong amide bond. The anti-IgE-aptamer modified G-FET showed selective electrical detection of IgE protein with a detection sensitivity of 0.29 nM. Furthermore, the dissociation constant between the aptamer and IgE was found to be 47 nM, indicating good IgE affinity of the graphene-based biosensor. Chen et al. developed a highly sensitive affinity sensor using rGO FETs decorated with gold nanoparticle antibody conjugates [21]. Immunoglobulin G (IgG) antibody was immobilized on the rGO surface through gold nanoparticles (AuNPs) and served as the specific receptor to capture IgG through antibodyantigen reaction (Fig. 9-4A). The lower detection limit of the rGO sensors was on the order of nanograms per milliliter, owing to the enhanced loading efficiency of antibody molecules through the introduction of Au nanoparticles (Fig. 9-4B). The negatively charged phosphate groups of nucleic acids (DNA and RNA) can lead to strong n-doping in graphene and subsequently change the electrical conductivity of the graphene transistor, which allows sensitive detection of nucleic acids by graphene-based affinity sensors (Fig. 9-4C) [22]. Chen and Li et al. immobilized single-stranded DNA (ss-DNA) receptors on the surface of graphene FETs. They showed that these graphene-based affinity sensors were able to detect the hybridization of target-DNAs to the receptor-DNAs with detection sensitivity of 0.01 nM [23]. In addition, the graphene-based affinity sensors showed single-base specificity. They further showed that the decoration of Au nanoparticles on graphene can effectively increase the upper detection limit from 10 to 500 nM.

9.1.3 Graphene Nanopores for the Detection of DNA Nanopore-based DNA analysis has been actively pursued in recent years. The basic principle of nanopore-based biosensing is to construct a pore structure with the dimension of several

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nanometers through a planar substrate (or making use of a natural nanopore structure), followed by the attachment to two fluid chambers filled with saline solutions. The solutions from the two chambers are connected only by the nanopore. When a voltage is applied between the two chambers, ions will be driven through the nanopore by the electric field, which thus generates a stable open-pore ionic current. The intensity of the transmembrane current is on the order of pA to nA. When negatively charged DNA is added into the chamber connected with the cathode, the DNA strand will be driven to translocate through the nanopore by electrophoresis. Since the electrophoresis mobility of DNA is lower than the mobility of the ions, the transmembrane conductivity will be decreased during DNA translocation, which produces a current blockage signal in the recorded transmembrane current and allows the detection of the DNA molecule. In principle, each nucleotide on the DNA strand can generate a specific current blockage, which could theoretically allow DNA sequencing. One advantage of nanopore-based DNA sequencing is that the sequencing process could be completed with even a single DNA

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molecule, which can thus eliminate the complicated amplification and labeling process. Nanopore-based DNA sequencing has been actively developed as the third generation gene sequencing techniques [24]. However, the thickness of traditional nanopore substrates, SiNx or SiO2, is usually from several to several tens of nanometers. When a DNA strand translocates through the nanopore, there would be more than one nucleotide inside the nanopore, which thus prevents the discrimination of a single nucleotide. Therefore, the optimal thickness of nanopores should be less than the distance between adjacent nucleotides in the DNA strand to achieve single nucleotide resolution. Graphene is a 2D nanomaterial with a thickness of single atomic layer and thus an ideal nanopore substrate for single nucleotide resolution (Fig. 9-5A) [2628]. In addition, the current through a graphene FET can be simultaneously measured with the transmembrane ionic current. Radenovic et al. showed (A) Graphene Constriction

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that the negative charges of the translocating DNA could change the carrier concentrations of a graphene nanoribbon, which thus led to spikes in both the transmembrane current and the drain current of the graphene transistor (Fig. 9-5B) [25]. However, the sizes of the nanopores through the graphene membrane are still so large that the translocation speed of DNA is beyond the bandwidth of the state-of-art current amplifiers. As a result, single-nucleotide discrimination and sequencing of DNA has not been achieved with graphene-based nanopores. Future studies are needed to precisely control the size of the nanopore to reduce the translocation speed of DNA and improve the resolution to single nucleotide.

9.1.4 Graphene-Based Tissue Engineering Tissue engineering has shown great promise to create biological alternatives for implants. In tissue engineering, three-dimensional (3D), porous scaffolds are required to accommodate cells and guide their growth and tissue regeneration. However, existing polymer scaffolds for tissue engineering usually suffer from the lack of electrical conductivity for cell stimulation. The integration of 2D graphene sheets into macroscopic, conductive 3D scaffolds can provide 3D cell growth microenvironments and synergistic guidance cues. Cheng et al. prepared a 3D porous graphene scaffold using nickel foams as sacrificial templates (Fig. 9-6A) [29]. The graphene sheets were seamlessly interconnected into a 3D flexible network, which led to both high surface area and good electrical conductivity of the scaffolds. The macroscopic, 3D graphene scaffolds had high porosity and specific surface area and could promote the adherence and growth of cells. For example, Dai, Tang, and Cheng et al. applied the 3D graphene scaffold as a novel scaffold for neural stem cells [30]. They showed that 3D graphene scaffold can enhance the differentiation of neural stem cells towards neurons and astrocytes. In addition, owing to the high electrical conductivity of the 3D graphene scaffolds, they were further used for efficient electrical stimulation of the neural stem cells (Fig. 9-6B). Sung et al. applied the 3D graphene scaffolds as material-derived cues for human mesenchymal stem cells. They showed that the 3D graphene scaffolds could maintain stem cell viability and promote spontaneous osteogenic differentiation [31].

FIGURE 9-6 Graphene scaffold for tissue engineering. (A) Morphology of a porous graphene foam [29]. (B) Growth of neuronal cells on the surface of a graphene foam [30].

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Graphene derivatives have also been used as additives in polymer matrix to increase their electrical conductivity and mechanical strength. For example, Murray and Wallace et al. prepared a conducting graphene/chitosan hydrogels [32]. Composites of graphene in a chitosanlactic acid matrix exhibited tunable swelling properties and excellent biocompatibility. The addition of graphene to the polymer matrix effectively improved both the mechanical strength and electrical conductivity of the hydrogels. Furthermore, they showed that 3D scaffolds based on the composites provided good adhesion for fibroblast cells. By the reduction of GO/polyacrylamide (PAAm) composite, Lee et al. synthesized a conductive r(GO/PAAm) hydrogel [33]. The r(GO/PAAm) hydrogel exhibited muscle tissue-like stiffness with a Young’s modulus of c.50 kPa. Compared to that of PAAm and unreduced GO/PAAm, the electrochemical impedance of r(GO/PAAm) was decreased by an order of magnitude. In addition, the conductive r(GO/PAAm) hydrogel could promote the proliferation and myogenic differentiation of myoblasts. They further demonstrated that electrical stimulation of the myoblast cells on the r(GO/PAAm) hydrogels significantly enhanced the myogenic gene expression.

9.2 Graphene Derivative-Based Functional Carriers Dispersed GO flakes, with a lateral dimension from several to several hundreds of nanometers, can be prepared on a large scale by chemical oxidation and exfoliation of natural graphite. GO is hydrophilic in nature as it contains extensive oxygen functionalities, such as hydroxyl, epoxy, and carboxy groups. GO can be reduced to rGO with partially restored π-conjugated domains through hydrazine treatment, thermal annealing, or microwave radiation. Both GO and rGO are photoluminescent with tunable spectra. They are more stable against photobleaching than organic fluorophores. In addition, GO and rGO can quench adsorbed fluorophores, including organic dyes and quantum dots. Thus GO can be applied in both fluorescent biosensing and bioimaging. GO and rGO can produce heat under laser irradiation, which makes them suitable as photothermal agents in cancer treatment. GO and rGO can be functionalized with various biomolecules and drugs through strong ππ stacking. In addition, the oxygen-containing groups on GO and rGO can serve as sites for chemical modification, which further allows their feasible functionalization through covalent bonds. The good dispersibility, high surface area, and ease of preparation and functionalization make GO and rGO attractive candidates in drug delivery.

9.2.1 Graphene Oxide-Based Fluorescent Biosensing Fluorescence resonance energy transfer (FRET) is a quantum process in which the excitation energy of a donor fluorophore is nonradiatively transferred to a nearby acceptor molecule via long-range dipole 2 dipole coupling. As a result, the fluorescence of the donor is quenched. FRET has been recognized as a sensitive and selective analytical technique in biosensing. In particular, GO has been shown to cause fluorescence quenching of various types

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of organic fluorescent molecules and quantum dots, owing to its delocalized π-conjugated domains. In 2009, Yang et al. reported a GO-based FRET platform for the sensitive detection of ssDNA and proteins [34]. Dye-labeled ssDNA molecules were absorbed on GO through strong ππ stacking, which then induced FRET between the dye-labeled ssDNA donor and GO. As a result, the fluorescence of the dye-labeled ssDNA was completely quenched. In the presence of the target ssDNA molecules, the dye-labeled ssDNA hybridized with the target ssDNA, which changed the configuration of the dye-labeled ssDNA and subsequently disabled its energy transfer to GO. Consequently, the fluorescence of the dye-labeled ssDNA was recovered, which allowed sensitive detection of the target ssDNA molecules. Li and Lin et al. applied GO-based FRET for in situ biomolecular probing in living cells (Fig. 9-7A) [35]. Dye-labeled ATP-aptamer was loaded on GO nanosheets to form a FRET pair. In the presence of ATP, the ATP-aptamer could target ATP to form a duplex configuration. As a result, the dye-labeled ATP-aptamer was released from the surface of GO, which led to fluorescence recovery (Fig. 9-7B). They further incubated the dye-labeled ATP-aptamer/GO complex with mice epithelial cells and observed significant fluorescent recovery of the dye-labeled ATPaptamer, indicating successful intracellular aptamer delivery and ATP probing in living cells. Jiang and Li et al. reported a GO-based FRET sensor for thrombin detection [37]. A dyelabeled aptamer was assembled on the GO surface to form a FRET pair. The addition of (A)

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thrombin led to the fluorescence recovery of the dye-labeled aptamer due to the formation of quadruplex-thrombin complexes. Seo et al. reported the detection of rotavirus by FRET quenching between GO sheets and AuNPs [36]. A rotavirus-specific antibody was chemically linked onto GO surface, and AuNPs linked with a secondary antibody were added to the system. The target rotavirus could bridge antibody-conjugated AuNPs with GO, which subsequently led to the fluorescence quenching of AuNPs (Fig. 9-7C).

9.2.2 Graphene Oxide-Based Bioimaging The photoluminescence (PL) in GO and rGO can be attributed to both their delocalized π-conjugated domains and also their edges/defects. The delocalized electrons of GO and rGO favor a rapid energy transfer, which enables a high temporal resolution of the photoluminescence signals. In addition, the PL spectra of GO and rGO depend on many factors, including their chemical structures and sizes. GO nanosheets with PL tunable from visible to infrared have been obtained. Combined with the high photostability and low toxicity of GO and rGO, they have been applied as optical probes in biological imaging. The application of the photoluminescent GO nanosheets for cellular imaging was first reported by Dai et al. [38]. GO nanosheets with lateral dimensions from 10 to 50 nm were covalently functionalized with polyethylene glycol (PEG) to improve their biocompatibility. The PEGylated GO was photoluminescent in the visible to IR regions. Rituxan, a B cellspecific anti-CD20 antibody, was further conjugated covalently onto the GO surface through the PEG linker. After culturing with lymphoma B cells and CD20 negative T-cells, the GO was found to selectively penetrate into the B cells (Fig. 9-8A). In addition, GO could be applied for in vivo tumor targeting and imaging. Liu et al. labeled the PEGylated GO with Cy7, a commonly used NIR fluorescent dye, to study the in vivo behaviors of GO nanosheets [39]. They showed that the small size and biocompatible PEG coating favored the enhanced permeability and retention (EPR) effect of GO nanosheets. In vivo fluorescence imaging revealed the high tumor uptake efficiency of GO nanosheets in several xenograft tumor mouse models (Fig. 9-8B). (A)

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FIGURE 9-8 Graphene oxide (GO)-based bioimaging. (A) Anti-CD20 conjugated GOPEG for in vitro imaging of CD20 positive Raji B cells (left) and CD20 negative T cells (right) [38]. (B) GOPEGCy7-based in vivo images of mice bearing 4T1, KB, and U87MG tumors [39].

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GO nanosheets can be conjugated with functional nanoparticles for bioimaging. For example, Zhang et al. attached Fe3O4 nanoparticles onto the surface of GO nanosheets for the magnetic resonance imaging (MRI) of HeLa cells [40]. Compared with Fe3O4 nanoparticles, the Fe3O4/GO complex showed dramatically enhanced MRI signal. In addition, Cai et al. prepared a conjugation of GO and 64Cu labeled CD105 antibody for in vivo positron emission tomography (PET) imaging to target 4T1 breast cancer tumors [41].

9.2.3 Graphene Oxide-Based Nanocarrier for Drug Delivery GO is an attractive candidate for efficient drug and gene delivery. Firstly, the chemistry structure of the GO nanosheets allows their easy functionalization with drugs and genes. The high surface area of GO nanosheets favors high loading efficiency of drugs and genes. Secondly, GO nanosheets can both effectively protect the drugs and genes from enzymatic degradation and also dramatically increase their in vivo circulation time. Thirdly, GO nanosheets can be conjugated with multifunctionalities, which allows targeted drug delivery and therapy. Dai et al. first reported the exploration of GO as novel and efficient nanocarrier for cancer drug delivery [38,42]. PEGylated GO nanosheets were conjugated with Rituxan to target the tumor tissues [38]. Doxorubicin (DOX), a water insoluble anticancer drug, was then loaded on the GO nanosheets through ππ stacking (Fig. 9-9A). The GOPEGRituxan/DOX was accumulated near the tumor tissues. Due to the acidic extracellular environment within the tumor tissues, the GOPEGRituxan/DOX complex slowly released DOX to the extracellular fluid, which resulted in high therapeutic efficacy for in vivo cancer treatment in a mouse model. Jing et al. further showed that the loading efficiency of DOX on GO could reach as (A)

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high as 200% [44]. Zhang et al. conjugated two cancer drugs, Camptothecin (CPT) and DOX, onto the GO surface through noncovalent interactions. The resulting CPT/DOX/GO showed remarkably higher cytotoxicity to cancer cells compared to GO loaded with either DOX or CPT only [45]. The success of gene therapy is largely dependent on the development of novel gene carriers. Recently, gene transfection into target cells using GO as a nanocarrier has been demonstrated by several groups. These results show that GO can effectively improve the transfection efficiency of DNA by both protecting them from nuclease degradation and enhancing their cellular uptake. Lin et al. showed that ss-DNA adsorbed on GO was effectively protected from enzymatic cleavage by DNase I [46]. Liu et al. prepared a complex of GO and polyethyleneimine (PEI), a cationic polymer. The GOPEI complex showed significantly reduced cellular toxicity compared to bare PEI. The positively charged GOPEI complex was then loaded with negatively charged plasmid DNA (pDNA) for the transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells (Fig. 9-9B) [43]. High EGFP expression was observed by using the GOPEI as the nanocarrier. The strong optical absorbance of GO in the near-infrared (NIR) region can be explored for in vivo photothermal therapy. Compared with AuNPs, GO has the advantages of easy and low-cost preparation and low cytotoxicity [47]. Liu et al. reported highly efficient tumor ablation in a mouse model after intravenous administration of GO and low-power NIR laser irradiation [39]. Zhong et al. conjugated both PEG and DOX onto the surface of GO. The synergetic effect of both chemical and photothermal treatment resulted in a greatly improved antitumor efficiency [48]. In addition, GO could be linked with photosensitizers to generate reactive oxygen species under light excitation, which resulted in an improved cancer cell photodynamic destruction effect [49].

9.3 Biosafety of Graphene The safety of graphene and its derivatives is of central importance for their biomedical applications. Due to the carbon-based structure, graphene and its derivatives were initially considered as biocompatible nanomaterials. However, a series of biosafety studies on graphene and its derivatives have indicated that their toxicity depends largely on their chemical structures, sizes, and surface functionalities. As a scaffold for tissue engineering, CVD-graphene has been shown to be a biocompatible substrate for cell adhesion, proliferation, and differentiation. For example, Pastorin and Özyilmazet et al. reported that CVD-graphene was a promising biocompatible scaffold for the proliferation of human mesenchymal stem cells (hMSCs) [50]. In addition, CVDgraphene substrates could accelerate the specific differentiation of hMSCs into bone cells. We note that, similar to many other inorganic nanomaterials, graphene cannot be easily biodegraded in vivo. The difficulty of degradation may bring long-term effects of graphene on the living organisms, which is thus a critical issue to be addressed before their clinical applications.

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Dispersed GO nanosheets could induce in vitro and in vivo toxicology. For example, GO nanosheets could pass through the cytomembrane to enter the cells and cause an increase of reactive oxygen species, which reduced the viability and even induced the apoptosis of cells. Dash et al. reported that the intravenously injected GO nanosheets were concentrated mainly in the lungs of mice and could cause pulmonary thromboembolism [51]. Furthermore, in vivo injection of GO nanosheets evoked strong aggregation response of human blood platelets [52]. The toxicity of GO depends on many factors including concentration [53], flake size [54], and surface functionalization. It has been shown that micrometerscale GO flakes could induce an even more severe inflammatory response than nanometerscale flakes [55]. Surface functionalization can also modify the toxicity of GO nanosheets. For example, Koyakutty et al. found that GO nanosheets aggregated on the cytomembrane of monkey kidney cells, which caused the destruction of cytoskeleton [56]. The surface of GO nanosheets was then carboxylated by nitric acid treatment. The carboxylated GO was shown to enter the cells without damaging the structure of the cell. As a result, the cytotoxicity threshold of GO was increased from 50 to 300 µg/mL [56]. PEG is widely used to increase the biocompatibilities and blood circulation of nanomaterials. Liu et al. intravenously injected GO and PEGylate GO into a mouse, respectively. They found that bare GO was quickly accumulated at the liver and spleen before being removed by the reticuloendothelial system (RES) [57]. On the other hand, PEGylate GO was greatly enhanced in the blood circulation.

9.4 Conclusions and Outlooks Graphene and its derivatives have demonstrated great potential in biomedical applications. The unique electrical properties of high-quality graphene, combined with its 2D structure, allow sensitive detection of both biomolecules and electrophysiological signals. Owing to the large surface area, good water dispersibility, and ease of preparation and functionalization, GO and rGO have been applied in bioimaging, fluorescence biosensing, drug delivery, and cancer therapy. Despite these promising developments, the biosafety of graphene and its derivatives needs to be systematically evaluated before clinical applications. Previous studies have shown that both size and surface functionalities play critical roles in regulating the biological behaviors and toxicity of graphene and its derivatives. In spite of this, there is a lack of a comprehensive understanding of the interactions of graphene and its derivatives with biological systems. In particular, the long-term toxicity of graphene and its derivatives needs to be investigated. Nevertheless, it is expected that the cooperative efforts from different areas, including chemistry, materials science, biology, and medicine, can result in synergistic progress in the biomedical applications of graphene and its derivatives.

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