Advances on graphene-based nanomaterials for biomedical applications

Accepted Manuscript Advancements of graphene-based nanomaterials in biomedicine

Ying Qu, Feng He, Chenggong Yu, Xuewu Liang, Dong Liang, Long Ma, Qiuqiong Zhang, Jiahui Lv, Jingde Wu PII: DOI: Reference:

S0928-4931(17)33059-X doi:10.1016/j.msec.2018.05.018 MSC 8573

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Materials Science & Engineering C

Received date: Revised date: Accepted date:

2 August 2017 26 March 2018 3 May 2018

Please cite this article as: Ying Qu, Feng He, Chenggong Yu, Xuewu Liang, Dong Liang, Long Ma, Qiuqiong Zhang, Jiahui Lv, Jingde Wu , Advancements of graphene-based nanomaterials in biomedicine. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi:10.1016/ j.msec.2018.05.018

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Advancements of graphene-based nanomaterials in biomedicine

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Ying Qu†, Feng He†, Chenggong Yu†, Xuewu Liang†, Dong Liang†, Long Ma‡, Qiuqiong Zhang†, Jiahui Lv†, Jingde Wu*

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† Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Science, Shandong University, Jinan, Shandong, 250012, China

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‡Department of Analytical Chemistry, the testing center of Shandong Bureau, Jinan, Shandong, 250014, China

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ABSTRACT Graphene-based nanomaterials, such as graphene oxide and reduced graphene oxide, have been attracting increasing attention in the field of biology and biomedicine over the past few years. Incorporation of these novel materials with drug, gene, photosensitizer and other cargos to construct novel delivery systems has witnessed rapid advance on the basis of their large surface area, distinct surface properties, excellent biocompatibility and pH sensitivity. Moreover, the inherent photothermal effect of these appealing materials enables them with the ability of killing targeting cells via a physical mechanism. Recently, more attentions have been attached to tissue engineering, including bone, neural, cardiac, cartilage, musculoskeletal, and skin/adipose tissue engineering, due to the outstanding mechanical strength, stiffness, electrical conductivity, various two-dimensional (2D) and three-dimensional (3D) morphologies of graphene-based nanomaterials. Herein, emerging applications of these nanomaterials in bio-imaging, drug/gene delivery, phototherapy, multimodality therapy and tissue engineering were comprehensively reviewed. Inevitably, the burgeon of this kind of novel materials leads to the endeavor to consider their safety so that this issue has been deeply discussed and summarized in our review. We hope that this review could offer an overall understanding of these nanomaterials for later in-depth investigations.

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1. Introduction

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KEYWORDS: graphene; physicochemical properties; biocompatibility; bio-imaging; delivery; phototherapy; tissue engineering

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Carbon is one of the essential elements on the earth. Nanocarbon opened a door to these novel nano-sized carbon allotropes, which basically contain three different dimensions, zero dimension (0D), one dimension (1D) and two dimension (2D). This is represented by fullerene, carbon nanotube, graphene respectively (Fig 1a,b,c). The crazy chasing for carbon nanomaterials began with the first discovery of the 0D fullerene (C60) in 1985 by Kroto et al.[1] Following that, Iijima brought 1D carbon nanotubes (CNTS) into the awareness of the scientific community in 1991.[2] In 2004, Andre Geim and Nosovelov successfully isolated single-layer graphene from graphite by mechanically cleaving a graphite crystal, pushing this kind of nanomaterials development to an exciting climax once again.[3] Their studies have confirmed that graphene exists in a 2D honeycomb-type lattice structure formed by a single sheet of sp2-bonded carbon atom. It is a milestone that firstly provided solid evidence to support the long existing graphene theory which can be traced back to the year of 1947.[4]

Figure 1. The structure of fullerene (a), single walled carbon nanotube (b), graphene (c), graphene oxide (d), carboxyl graphene oxide (e) and reduced graphene oxide (f).

Since then, the potential applications of graphene-based nanomaterials in biomedical field have been intensively explored for the unique physicochemical properties associated with these materials. Specially, the progress has been made towards drug/gene delivery,[5, 6] phototherapy,[7, 8] cellular growth and differentiation,[9, 10] biosensors,[11-13] bio-imaging,[14,

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the same plane,[28] while opposite to diamond, the interlayer bonding via weak Van der Waals forces makes it a soft material.[29] Another significant merit is its large surface area on both sides of the sheet (2630 m2/g) that endows graphene with the ability to absorb or bind more cargos.[30] Compared with the hydrophobic nature of pristine graphene that often causes irreversible aggregation[31] and protein adsorption[32], GO contains both hydrophobic and hydrophilic regions that confer it with an amphiphilic characteristic. On hydrophobic flat region, the π-π conjugate system of its surface gives it the capacity to load chemical drugs, genes, photosensitizers and other molecules via π-π interaction or electrostatic attraction. The hydrophilic region allows for functionalization. However, the presence of functional groups creates high defect density, which could diminish the electrical conductivity, mechanical and optical properties.[33] In contrast with GO, GO-COOH has more anchor sites for functionalization, while RGO has more flat region for loading cargos.

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15] cancer or disease detection and therapeutics[16-19], and tissue engineering[20, 21] (Fig 2). To date, these materials have showed expanded potential in diagnose and treatment of various diseases,[10, 22-25] especially malignant tumor, a major killer of human health. Although emerging evidences indicate that graphene-based materials have promising potential in biomedical field, concerns have been raised on its potential danger to human and environment.[26] At present, existing results on toxicity evaluation remain controversial, which indicates that there is still a long way before these novel materials being applied in clinic, in despite of massive exciting results. This review is focused on summarizing the physicochemical properties, preclinical biocompatibility, and applications of these graphene-based nanomaterials in biomedical field, together with envisioned challenges and future perspectives.

2.2 Biocompatibility

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To retain single sheets in different matrices is crucial for graphene-based nanomaterials. However, the monolayer graphene trends to re-aggregate in solvents, primarily due to the existence of the intermolecular attractive forces, principally including the Van der Waals forces, π-π stacking, electrostatic interaction, and hydrogen bond. Therefore, the sufficient dispersion of graphene-based nanomaterials in various solvents is a prerequisite for their further application.[34] The active groups on the surface of GO derivatives confer an amphiphilic nature to them, allowing GO derivatives to possess good dispersion in a plenty of solvents. However, GO still tends to form aggregates in solutions with high concentration of salts or protein,[35] like physiological saline and serum (Fig 3a). To address the aggregation issue, surface coating by structural modifications has been adopted. To this end, two approaches, covalent method and non-covalent method have been devised to modify the surface. The covalent method refers to chemical reactions, while the non-covalent method is mainly achieved via Van der Waals forces, π-π interactions or electrostatic attraction. But, neither method is satisfactory. Non-covalent bond typically cannot hold the material stable in the prolonged circulation and the loading capacity is generally not optimal. Though covalent bonds seem more stable than non-covalent bond, the number of different functional groups incorporated cannot be determined precisely. Besides, surface coating by covalent method may not represent an ideal strategy to modify RGO directly, since the functional groups are minimal on the edge of RGO after chemical reduction.

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Figure 2. The primary application of graphene-based nanomaterials in biomedicine.

2. Properties of graphene-based nanomaterials

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A variety of functionalized forms of graphene-based nanomaterials have been developed and well-studied, mainly including graphene, graphene oxide (GO), carboxyl graphene (GO-COOH) and reduced graphene oxide (RGO) (Fig 1d,e,f, respectively). These novel nanomaterials have some unique properties, such as large surface area, distinct surface properties, improved biocompatibility, photothermal effect, pH sensitivity, etc. We will discuss these properties in detail in the following sub-sections.

2.1 Surface properties The carbon atoms of graphene, existing in the form of sp 2 hybrid, constitute a huge π-π conjugate system which the electrons are freely moving in. This is responsible for the perfect planer construction, strong electrical conductivity (ballistic electron transport with carrier mobility of 10 000 cm2V-1S1 )[27] and mechanical properties (~1TPa)[28] of graphene. Single-layer graphene possesses a thickness of ~0.34nm.[3] Its hardness is higher than diamond due to strong C-C bonding in

2.3 Photothermal property Upon irradiation with near infrared ray, the photon energy is converted into heat via non-radiative decay transitions, which could be used to kill cancer cells for PTT.[36] Meanwhile, the non-covalent bond interactions on the surface of graphenebased nanomaterials diminish, due to the absorption of light, increased temperature and atomic vibrations. Hence, graphene-based nanomaterials not only could be directly applied to photothermal therapy, but also could be used to rapidly release the cargos from the surface via near infrared ray irradiation. Compared with GO, RGO has been proven to show

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3. Preclinical biocompatibility study of graphenebased nanomaterials

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3.1.2 Hemocompatibility

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Biocompatibility refers to the ability of materials to interact with cells, tissues, or body without causing damage.[42] Extensive studies have been conducted to illuminate the preclinical biocompatibility of graphene-based nanomaterials in vitro and in vivo, which is critical to identify suitable candidates for clinical applications.

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2.4 PH sensitivity The surface properties of graphene-based nanomaterials can be influenced by pH change. Studies by our group and others have proved that GO sheets could disperse at pH values ranging from 3 to 12 and are most stable at pH 7 or 8.[38] Tumor tissues are generally more acidic (pH~6.8) environment than normal cells (pH 7.4).[39] In lower pH, the hydrogen-bonding interaction between drug and GO is weakened for the protonation. This property of GO has been incorporated into the design of a pH-triggered drug release system. Collectively, graphene-based nanomaterials with special physicochemical properties appear to be promising nanomaterials to be applied in biomedicine. However, some limitations still exist, such as the heterogeneous size, the unknown mechanism of clearance and knowledge gap in GO-related biomedical research. Further investigations are needed to address above problems.[40, 41]

The mechanisms of the antimicrobial activities of graphene materials were summarized by Zou and coworkers, including Ⅰ) the action of sharp edges; Ⅱ) oxidative stress mediated with or without the production of ROS; Ⅲ) wrapping or trapping of bacterial membranes; Ⅳ) extraction of lipid bilayers; Ⅴ) interference of protein−protein interactions; 6) “self-killing” effect.[46] At present, the antibacterial activity have been intensively investigated in wound sites to prevent infections. For toxicity to eukaryotic cells, multiple mechanisms have been considered to lead to cellular toxicity, including oxidative stress, genotoxicity, autophagy, apoptosis, immune responses etc.[47] Pristine graphene has been reported to cause high oxidative stress by accumulation on the cell membrane. The hydrophobic nature of graphene may be responsible for cellular toxicity by interrupting the hydrophobic proteinprotein interaction in the membrane. Graphene enter the hydrophobic interface to separate two functional proteins to disrupt the cell’s metabolism or even lead to the cell’s mortality.[48] RGO possesses greater cellular toxicity than GO and their biological and molecular mechanisms are different, which is attributed to the different surface oxidation status.[47] However, functionalized graphene-based nanomaterials show remarkably reduced cellular toxicity as mentioned above.

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preferable conductive and optical absorbance,[37] which might be explained by its broader flat region that resembles the structure and properties of graphene.

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3.1 Biocompatibility in vitro

In vitro biocompatibility tests generally involve cytotoxicity to prokaryotic cells and eukaryotic cells, hemocompatibility, as well as inflammatory responses.

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3.1.1 Cytotoxicity

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Uncoated graphene-based nanomaterials have a dosedependent toxicity in cell assay and can easily assemble in physiological saline. The toxicity evaluation of these nanomaterials in vitro is mainly tested in prokaryotic cells and eukaryotic cells. A large number of graphene-based nanocomposites have been proved to exhibit toxicity to both prokaryotic and eukaryotic cells. On the one hand, graphene-based nanomaterials can be used to achieve promising antibacterial activity against prokaryotic cells. On the other hand, the rational surface-modified ones can exhibit reduced toxicity and improved biocompatibility to eukaryotic cells. For toxicity to prokaryotic cells, some studies suggested graphene and GO had noticeable antimicrobial effect by cell membrane damage.[43] In another report, graphene derivatives have been sufficiently proved to enhance the antibacterial activity of metal and metal oxide nanostructures by oxidative stress induction and membrane disruption.[44] Recently, Kim et al. prepared effective antibacterial surface consisting of GO, MoS2, which showed enhanced antimicrobial effects toward the Gram-negative bacteria Escherichia coli with increased glutathione oxidation capacity and partial conductivity.[45]

Hemocompatibility investigation is an important toxicity assessment for biomedical application. Red blood cells (RBCs) are one of the primary interaction sites. During blood transfusion, hemolysis may cause organ failure and even patient death.[49] In generally, fresh blood collected from healthy rabbits is applied for hemolytic analysis. Both graphene and graphene oxide exhibit dose-dependent hemolytic activity in RBC cells. Comparing to graphene sheets, GO, with higher surface oxygen content, has enhanced hemolysis due to the electrostatic interaction between the GO and RBC outer membrane. GO shows greater hemolytic activity with smaller size.[50] However, too larger-sized GO can retain and cause inflammatory responses for not being easily swallowed by macrophages. Proper surface modification could improve the hemocompatibility of GO. For instance, Chitosan grafted GO showed eliminated hemolysis by masking the electrostatic interaction.[51] bovine serum albumin (BSA) modified GO nanosheets (GO-g-BSA), constructed by Cai and coworkers, exhibited obviously improved hemolysis ratio (lower than 0.2%) and no visible hemoglobin release.[52]

3.1.3 Inflammatory responses Macrophages could engulf foreign substances through phagocytosis to play an important role in non-specific immune defense, but they also establish a barrier against GO. So GO could be eliminated or induce inflammatory responses before reaching the target. It has been reported that micro-sized GO could cause serous inflammatory responses proved by remarkable increase of IL-6, TNF-α, MCP-1 IL-12, and IFN-γ.[53] Besides, Ma et al. proposed that larger GO could induce more inflammatory cytokines by interacting with toll-like receptors and activating NF-kB pathway.[54] According to the study of Kiew and coworkers, nanomaterials with more hydrophilic surface and controlled size at approximately 150nm could

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3.2.3 Metabolism As aforementioned, clearance of functionalized graphenebased nanomaterials is primarily through renal and fecal excretion. However, whether these novel nanomaterials could be degraded is still controversial. Graphene has been applied in the field of biomedicine in the past decade to be deemed biodegradable. To support this property, studies by Girish et al. provided clear evidence of in vivo biodegradation by changes of D and G bonds in confocal Raman imaging.[40] Although GO could gradually degraded through enzyme oxidization by horseradish peroxidase (HRP), both PEG and bovine serum albumin (BSA) coated GO and RGO are resistant to HRPinduced degradation except in the presence of a cleavable disulfide bond (Fig 3c).[63]

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3.2 Biocompatibility in vivo In this section, the pharmacokinetics (including absorption, distribution, metabolism and excretion) were summarized using mice or rats as animal model, and the in vivo toxicity of graphene-based nanomaterials to zebrafish, mice, rats, rabbits and canines was deeply discussed. 3.2.1 Absorption Research on the absorption of graphene-based nanomaterials is mainly based on preclinical animal studies using mice or rats as animal model. Different drug administration routes, such as inhalation, oral feeding, intravenous injection and intraperitoneal (i.p.) injection for rats give rise to different absorption routes. Studies by Li et al. found that NGO was initially retained in the lung issue after inhalation, as evidenced by radioisotope tracing and morphological observation. Then NGO passed through the air-blood barrier into blood and reached other organs.[55] In addition, macrophage uptake of graphene was observed after inhalation.[56] After oral administration, limited absorption of graphene-based nanomaterials by the digestive system was reported, evidenced by no significant tissue uptake of 125I labeled PEGylated GO derivatives. After intraperitoneal (i.p.) injection, PEGylated GO derivatives highly accumulated in reticuloendothelial system (RES), including liver and spleen. Notably, it was observed that RGO-PEG with larger size showed nearly 2-folds RES uptake compared to these with smaller size, suggesting that size of nanomaterials could be an important factor in determining adsorption after i.p. administration.[57] The uptake mechanism of 2D nanomaterials is size and dose dependent. Small sized protein-coated graphene oxide (PCGO) enter cells mainly through clathrin-mediated endocytosis (CME), while large nanosheets enter cells through both CME and phagocytosis (Fig 3b).[58] After intravenous (i.v.) administration, GO without surface modification predominantly deposited in the lungs.[59] However, NGS-PEG mainly accumulated in the reticuloendothelial system (RES).[60] 3.2.2 Distribution and excretion The first study on the long term distribution of PEGylated graphene in mice was reported by Yang et al. using radio labeled substrate.[60] Their results demonstrated that 125I labeled NGS-PEG mainly accumulated in the RES after i.v. administration, such as liver and spleen, followed by clearance through renal and fecal excretion. To rule out the possibility that the observed excretion of NGS-PEG was not induced by the degradation of the radiolabels, they further conducted immunohistochemistry (IHC) studies of the liver tissue by haematoxylin and eosin (H&E) staining (Fig 3d,e,f ), which was in agreement with the distribution results based on radioactivity measurements. In addition, no apparent toxicity was observed at a dose of 20 mg/kg as evidenced by the biochemical analysis, hematological analysis, and histological examinations. Studies by Li et al. indicated that NGO primarily accumulated in and subsequently cleared from the lung tissue in

mice after inhalation.[55] The effects of PEG coating on the distribution and toxicity of NGO were also examined by the same group in mice. NGO mainly retained in the liver, lung and spleen, mainly due to NGO aggregation. In contrast, PEG coated NGO exhibited reduced retention and better clearance in these organs. PEGylation may also alleviate NGO-induced acute and chronic toxicity, suggested by Yang et al.[61] Studies by Lu et al. indicated that PEG-GO nanoribbons accumulated in the liver, then eliminated by renal excretion, thus echoing the results of Li’s study. [62] Despite the explosive studies on excretion as mentioned above, the underlying excretion mechanism is still uncovered.

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avoid being recognized by macrophage via weakening the opsonin–protein interaction.[41] Therefore, functionalized NGOs seem to be suitable candidates for avoiding inflammatory responses.

Figure 3. (a) Phenomenon of GO, nGO-PEG, RGO, RGO-PEG, nRGO, nRGO-PEG in water, saline and serum. Reproduced from ref [64]. Copyright (2012) Biomaterials. (b)Schematic illustration of cell uptake of PCGO. Reproduced from ref [58]. Copyright (2012) ACS Appl Mater Interfaces. (c) The biodistribution of 131I labeled GO and GO-SS-PEG in Balb/C mice. Reproduced from ref [63]. Copyright (2014) Small. H&E stained liver slice from control mice (d) and mice injected with NGS-PEG

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3.2.4 Toxicity in vivo

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For the toxicity of graphene-based nanomaterials to Zebrafish, an intermediate model, Jeong and coworkers[65] proposed that PEGylation attenuated gross morphological defects of zebrafish embryos caused by NGO, while Alexa568 conjugated, PEGylated NGO (NGO-A568) still caused angiogenic defects to the similar degree as NGO did using zebrafish embryos, suggesting that the different developmental processes had different toxic effect. However, no obvious toxicity was observed by Chen et al. when zebrafish was exposed to 50 mg/L GO for 14 days, but they detected a number of cellular alterations by histological analysis.[66] Kim and coworkers did a 1-, 28- and 90-day graphene inhalation toxicity study with male Sprague-Dawley rats, revealing no dose-dependent effects were recorded for the body or organ weights at 28-day post-exposure and even no distinct lung pathology was observed at 90-day post-exposure.[56] Yang and his coworkers[57] uncovered that no obvious in vivo toxicity caused by PEG-NGO was observed for 3 months in mice in different administration routes, including intravenous injection, intraperitoneal injection or oral feeding. It was reported that graphene or graphene oxide without further surface modification would cause severe pulmonary inflammation after inhalation.[67] Studies by Meng et al. indicated GO easily induced strong oxidative stress and inflammatory reaction in injection site of mice when used as vaccine carriers and adjuvants. However, once decorated by antioxidant Carnosine (Car), no symptoms such as allergic response, raised surface temperatures, inflammatory redness swelling, physiological anomalies of blood, or obvious weight changes were observed. [68] Further investigation about the influence of graphene-based nanomaterials on the reproduction provided evidence to support the safety of these materials. Xu et al. demonstrated that no apparent toxicity in the reproductive system of female mice was observed after treatment with RGO before fertilization (30~35 days before pregnancy).[69] The studies by Liang and coworkers also supported this notion as no apparent toxicity was observed for nano-sized graphene oxide on the reproductive system of male mice.[70] However, it does not mean that these nanomaterials are safe enough for productive system even in a lower concentration, thus more in-depth research is needed. In another study, the intraocular biocompatibility of microsized GO (~1μm) was explored by Yan et al. After being injected with 0.3 mg GO, the rabbit eyes were still clear with no inflammatory responses for 49 days, but GO might have diffused to the vitreous region of the eye.[71] Recently, more attention has been focused on applications of graphene-based nanomaterials in tissue engineering. Zhang and coworkers prepared graphene-reinforced nanohydroxyapatite/polyamide66 (nHA/PA66) bone screws implanted in canine femoral condyles. They revealed no obvious inflammatory reaction was observed in soft tissues and no obvious dam-

age to the liver, spleen, kidney, brain, and other major organs.[72] In conclusion, the intrinsic physicochemical properties of graphene-based nanomaterials, such as surface functional groups, charges, coating, sizes, and structural defects of graphene, may play essential roles in determining cellular biocompatibility.[73] It has been reported that smaller particle size and higher oxidation are beneficial to improve biocompatibility.[74] Besides, functionalization of these nanomaterials with macromolecules, such as PEG,[63, 75] bovine serum albumin,[63] dextran,[76] serum protein,[77] and chitosan[78] could help improve the biocompatibility and attenuate cytotoxicity. This could be the result of the change in the sheet size, thickness or nonspecific binding, thus leading to better dispersion and improved stability. Besides, the direct interactions between the cell membrane and GO are attenuated by modification, leading to a mitigated cytotoxicity caused by high protein adsorption of GO.[79] Furthermore, surface modification and functionalization could promote cellular growth and differentiation,[80] which is widely used in tissue engineering. Till now, biocompatibility and toxicity evaluation are not consistent. The opposite results may be caused by a great diversity of factors such as the differences between different experiment models, different experimental environment and condition, graphene-based carriers with different properties, etc. In despite of these different findings as mentioned above, most of the results agree that the toxicity in vitro and in vivo could be controlled, and with small size [81] and reasonable surface functionalization, these nanomaterials appear to be less harmful and possess excellent biocompatibility. And the currently available results support further development of these nanomaterials in clinical studies. Furthermore, long-term toxicity studies of these nanomaterials are warranted to add confidence to further develop the graphene-based nanomaterials in biomedical field.

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for 3d (e) and 20d (f). Reproduced from ref [60]. Copyright (2011) ACS Nano.

4. Application in biomedical field 4.1 Bio-imaging Bio-imaging allows to observe and study the biological processes from cellular and subcellular levels to animals, which plays critical roles in both research and clinical practice.[82] Graphene-based nanomaterials have been extensively explored for potential applications in various imaging techniques, including radionuclide-based imaging (PET and SPECT), photoacoustic imaging (PAI), computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography (US), optical imaging and multimodality imaging.[83] Optical imaging could provide detailed images of organs, tissues, cells and even molecules, using the special properties of photons. Graphene has been explored for optical imaging, including FL imaging, two-photon FL imaging (TPFI) and Raman imaging. Radionuclide-based imaging exhibits excellent sensitivity and limitless penetration depth by accurately tracking the labeled substances in vivo in a quantitative method.[14] PAI possesses high spatial resolution even at great depths since photoacoustic waves are much less scattered (2~3 orders of magnitude) than

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GO was absorbed onto the microcapsule surface via electrostatic LbL technique. The obtained microcapsules could serve as a contrast agent to simultaneously enhance US imaging and X-ray CT imaging both in vitro and in vivo. This synergetic US/CT imaging could not only accurately identify the size and location of the tumor but also guide and monitor the photothermal therapy.[91] Studies by Yue et al. described a pHresponsive FePt-based multifunctional theranostic agent for dual MRI/CT imaging and in situ cancer inhibition. [92] This FePt-DMSA/GO-PEG-FA composite could target tumor cells and the decomposition of FePt gave rise to T2-weighted MRI signal and increased ROS signal, enabling intuitively expression of Fe release in tumor cells. Zhang et al. established GO/bismuth selenide/polyvinylpyrrolidone nanocomposites through direct deposition of Bi2Se3 NPs on GO in the presence of PVP, which could serve as an efficient bimodal contrast agent to simultaneously enhance X-ray CT imaging and PAI imaging in vivo.[93] In summary, Graphene could play different roles in bioimaging 1) as imaging contrast agents; 2) as carriers; 3) as fluorescence quenchers; 4) as wrapping materials; 5) as building blocks.[14] This powerful technique exhibits great potential in biomedicine, which can not only detect and characterize disease but also rapidly monitor the treatment response.

4.2 Drug/gene delivery

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optical waves in biological tissue.[84] MRI provides highresolution anatomical images and excellent soft-tissue contrast by a non-invasive and non-ionizing method. Complementary anatomical information can be obtained by computed tomography (CT). Each imaging modality has its own advantages and limitations. Multimodal imaging has emerged as a superior modality because of its capability of overcoming the limitations of single imaging modality. Ions of paramagnetic metals functionalized GOs/rGOs are suitable for MRI. Clinically used gadolinium diethylene triamine pentaacetate (Gd-DTPA) can detect tumors with shortened longitudinal relaxation times (T1) of water proton and enhanced bright positive signal in MRI. However, these chelates also have some restrictions, like non-specificity and rapid renal clearance. To address this issue, Gadolinium diethylene triamine pentaacetate (Gd-DTPA) and prostate stem cell antigen (PSCA) monoclonal antibody (mAb) dual functionalized dendrimer-GO nanocomplex (GO-DEN(Gd-DTPA)-mAb) have been constructed and proven to show reliable MRI performance.[85] Based on the luminescent properties of graphene oxide, especially tailormade controlling emission colors, chemical oxidative strategy was explored for tuning fluorescence of GO nanosheets from brown to yellow, green, and cyan without changing excitation wavelength. This strategy achieved multicolor cellular imaging with enhanced the fluorescent quantum yields.[86] Photoacoustic imaging (PAI) is a powerful preclinical diagnostic tool with a high resolution. However, the poor tissue penetration upon NIR irradiation limited their clinical application. Most of fluorescent dyes are not suitable in the clinical studies for their toxicity, especially to liver and kidney, [87] but this problem could be settled by the delivery of suitable carrier. The rGOs or dyes/PSs grafted GOs have been developed for PAI, due to the strong optical absorbance in the NIR region. [14] Toumia and coworkers fabricated a hybrid system by tethering pristine graphene to poly (vinyl alcohol) based microbubbles (PVA MBs), achieving enhanced photoacoustic signals.[88] Radionuclide-labeled GOs are applied for PET/SPECT imaging. Cornelissen et al. explored the use of anti-HER2 antibody (trastuzumab)111 conjugated NGO, radiolabeled with In-benzyldiethylenetriaminepentaacetic acid (BnDTPA) via π-π stacking. Tumor accumulation of 111In-NGO-Tz was visualized clearly using SPECT imaging.[30] Multimodal imaging is achieved in one platform by integrating other materials or molecules with specific properties onto GO. Chen et al. fabricated PEGylated RGO nanocomplex with indocyamine green (ICG) as a new type of imaging agent, combing fluorescence and photoacoustic dual-modality.[89] This RNGO-PEG/ICG nanocomposite exhibited lower toxicity, improved stability, prolonged circulation time, superior passive targeting capability and stronger imaging signals with no obvious accumulation, exhibiting the potential for diagnosis and imaged-guided therapy. In another report, [90] reduced graphene oxide–iron oxide quantum dots (QDs) were synthesized by Justin and coworkers, allowing for fluorescent and MRI dual-modality imaging without using an additional fluorescent dye. A multifunctional microcapsule was synthesized by Jin and coworkers. They integrated Au NPs with PLA microcapsules through a double-microemulsion method, and then

Chemotherapy is an essential therapeutic approach to manage a variety of human diseases but this approach suffers from poor pharmacokinetic and serous side-effects. Additionally, multi-drug resistance (MDR) caused by over-expression of Pgp, is another main obstacle for successful chemotherapy.[94] To address these issues, suitable drug delivery system (DDS) can be designed to alter the pharmacokinetics (PK), biodistribution (BD) of associated drug apparently and reverse drug resistance of tumor cells. [95, 96] One of the key factors in constructing DDS is the selection of an appropriate carrier. Graphene-based nanomaterials have been considered to be one of the optimal choices, which can be engineered to integrate multiple functions in a single system.[97] Accurate targeted therapy can reduce damage to normal cells or tissues in anticancer therapy. Two methods, active targeting and passive targeting, have been devised to achieve this aim. Passive targeting could lead to low drug concentration in targeting cells due to the leakage and inactivation of drug during the prolonged circulation. Furthermore, the ionic interaction between the electronegative cell membrane and electropositive nanoparticles may cause toxicity and off-target effect in some cases.[98] In contrast, active targeting could achieve enhanced therapeutic effects and reduced side effects by the interactions between ligand and corresponding receptor. Modified GO, can deliver the loaded drug to target cells with excellent biocompatibility, low toxicity and specific targeting. This could not only improve the therapeutic effects, but also achieve real-time imaging of therapeutic response. Some nanomaterial based systems solely rely on passive targeting to reach tumors by the enhanced permeability and retention (EPR) effect.[95] For instance, Xu et al. manifested that PEG-GO loaded with PTX quickly entered human lung cancer A549 and human breast cancer MCF-7 cells and showed improved

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mal conversion of RGO gave rise to the abscission of RGO from the MSN-C18 surface and achieved the light-responsive DOX release.[104] Dihydroartemisinin (DHA) and transferrin (Tf) dual-dressed nano-GO (DHA-GO-Tf) was also reported.[105] Tf played two critical roles in this example, targeting ligand and ferric ion carrier. Results suggested complete tumor cure with no obvious side effects. Recently, the “energy molecule”, ATP has been employed as a trigger for controlled drug release, on the basis of the higher intracellular ATP concentration (1~10 mM) than extracellular level (<5 mM). In one study, GO/DNA1 and GO/DNA2 were linked by ATP apatamer to form aggregates upon hybridization for ATP-responsive on-demand DOX release, as shown in Fig 5.[106]

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cytotoxicity.[99] Active-targeting nanoparticle systems employ receptor-mediated endocytosis to achieve targeted delivery. For example, dendrimers capped with amino groups (DEN) were grafted to GO and this product was subsequently functionalized with gadolinium diethylene triamine pentaacetate (Gd-DTPA) and prostate stem cell antigen (PSCA) monoclonal antibody (mAb). The formulated GO-DEN (GdDTPA)-mAb nanoparticles were then employed to simultaneously achieve imaging diagnosis and chemotherapy in prostate cancers. Data showed that as-prepared nanoparticles exhibited specific targeting capability to PSCA over-expressed tumor cells, enhanced cytotoxicity, negligible hemolytic activity, and no overall toxic effects in mice.[85] Another example was from the studies by Tian et al.[100] The FA/CPT/Pep/GO complexes were engineered by conjugating PEGylated folate and fluorescein-labeled peptide to graphene oxide, showing high therapeutic efficiency and high selectivity to cancer cells, as shown in Fig 4. Recently, PEGylated GO loaded with PTX and indocyanine green (ICG) has also been reported.[101]

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Figure 4. Schematic illustration for the interaction of FA/CPT/Pep/GO nanocomplexes with targeted cells.

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The graphene-based nanomaterials also provide a new platform to realize controlled release by establishing some smart structures that are responsive to extra stimuli, like pH, light, heat, magnetic fields, etc.[39] Novel carriers, like GO-CMCFI-HA and GO-DEX-Apt have been constructed to release chemotherapeutic agents in a pH-sensitive way.[78, 102] In the hydrogen-bonding interaction between –OH or –NH2 groups in chemical drugs and the –OH and –COOH groups on GO is the strongest at the neutral condition, not beneficial for drug release. While in acidic environment, part of the hydrogen bond is broken for protonation, leading to a pH-triggered drug release. With regard to magnetic field responsive drug release, Ma and coworkers designed PEGylated reduced graphene oxide-iron oxide nanoparticle (RGO-IONP-PEG), capable of magnetically targeted DOX delivery. It was found that under magnetic field, cancer cells showed more DOX uptake than these far away from magnetic field.[103] Besides, He et al. successfully engineered MSN-C18-RGO, based on noncovalent assembly between RGO and alkyl-grafted functional mesoporous silica (MSN-C18) to load DOX. The photother-

Figure 5. Schematic illustration of ATP-responsive DNA-GO for controlled drug delivery. Reproduced from ref[106]. Copyright (2015) Biomaterials.

In generally, anti-tumor drugs are loaded on carriers via physical methods, while some drugs with no extended π-π structure like PTX, are selected to link to the surface of GO via chemical bond. Nano-system GO-PEG-PTX, in which anti-tumor drug PTX was conjugated to PEG by covalent bond, could be hydrolyzed by both chemical and enzymatic pathways with prolonged blood circulation time as well as high tumor-targeting and -suppressing efficacy.[75, 107] All these drug systems seem to have broad prospects to be applied in clinic as evidenced by these comprehensive experiment results. Gene therapy, using nuclei acids to regulate, replace, repair, add or delete a fraction of responsible gene sequence, has made significant progress in recent years. The therapeutically active nucleic acids include small-interfering ribonucleic acid (siRNA), short hair-pin ribonucleic acid (shRNA), microribonucleic acid (miRNA), antisense oligonucleotides (ODNs), plasmid DNA (pDNA) and RNA/DNA aptamers.[108] So far, the issue of gene delivery by employing viral or non-viral vectors remains a key bottleneck for gene therapy. Viral vectors with high transfection efficiency have been hampered for poor safety. Considering the merits of reduced toxicity, low immunogenicity and low carcinogenicity, non-viral vectors have been intensively studied. However, low transfection efficiency

ACCEPTED MANUSCRIPT GO-PEG-PEI (GPP). In addition, RGPP had remarkably enhanced transfection efficiency under NIR laser irradiation.[116] Recently, Wang et al.[117] successfully designed a plasmid delivery system GO-PEI-PEG-FA (folic acid)/si-Stat3 for treating hepatocellular carcinoma by continuous covalent functionalization of GO, PEI, PEG and FA, followed by loading si-Stat3 via electrostatic adsorption. Their studies indicated that the transfection efficiency was relatively high with no obvious toxicity When PEI accounted for 61% in the complex and the nanostructure varied from an intercalated layer structure to a fully exfoliated structure with the increasing of PEI. Polyamidoamine (PAMAM) dendrimer is biodegradable cationic and highly branched spherical polymer with a peptide bond backbone. In one study, Liu et al. established PAMAM dendrimer and oleic acid dual-grafted NGO.[118] Oleic acid is a nature product with high affinity to cellular membrane to induce membrane destabilization. According to their study, GO-oleate-PAMAM showed good biocompatibility, low toxicity and remarkably improved gene transfection efficiency (18.3%), compared with that by ultrasonicated graphene (1.4%) and the GO-PAMAM (7.0%). PAMAM dendrimer grafted NGO, established by Sarkar and coworkers, also exhibited higher transfection efficiency in HeLa cells (51%) than that by polyethyleneimine (27%) and Lipofectamine 2000 (47%).[5] Furthermore, Wang et al.[119] demonstrated PAMAM dendrimer and polyethylene glycol (PEG) dual-functionalized nanographene oxide conjugate (NGO-PEG-dendrimer) could efficiently deliver anti-miR-21 into non-small-cell lung cancer cells, resulting in a stronger inhibition of cell migration and invasion than dendrimer or Lipo2000 transfection did. In addition, Ren et al. successfully designed poly-L-lysine and Arg-Gly-Asp-Ser functionalized graphene oxide to deliver anti-VEGF siRNA with active tumor target. It was worth mentioning that Cy-3 labeled siRNA was observed to assemble in tumor tissues in vivo via fluorescence imaging, and consequently the gene expression of VEGF was down-regulated, achieving 51.74% tumor inhibitory rate at anti-VEGF siRNA dose of 0.3 mg·kg-1.[120] Overall, functionalized graphene oxides could be harnessed for gene delivery with enhanced transfection efficiency and reduced toxicity.

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Figure 6. Schematic illustration of the mechanism of utilizing modified GO as siRNA carriers to kill cancer cells.

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and poor transgene expression limit their further application in clinic.[109] Hence, some barriers must be overcome for the success of gene therapy, such as cellular uptake, endosomal escape, release from vectors, etc. Some non-viral vectors have gained significant attentions in gene delivery, including polymers, liquid, inorganic nanomaterials, etc. Recently, Integration of graphene-based nanomaterials with chitosan, polyetherimide, polyamidoamine dendrimer, and some other polymers have been extensively investigated. The mechanism of siRNA in killing cancer cells utilizing modified GO as carriers is briefly described in Fig 6.

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Chitosan, a biodegradable, biocompatible and mucoadhesive cationic polysaccharide with low cytotoxicity, has been successfully employed for in vitro and in vivo gene delivery application as a polymeric carrier. In one study, the prepared CS-grafted GO (GO-CS), consisting of 64wt.% CS to stable plasmid DNA, exhibited increased transfection efficiency. Incorporation of CS also improved the stability, solubility and biocompatibility of GO.[110] Wang and coworkers[111] constructed a chitosan functionalized magnetic graphene (CMG) nanoparticle platform, which successfully delivered DOX and DNA to implanted tumors in mice. However, the poor buffering capacity for endosomal escape is the major limitation.[112] Polyetherimide (PEI) shows strong binding to nucleic acid, effective uptake by cell and endosome release of gene. However, PEI, especially that with high molecular weight, possesses high toxicity and poor compatibility, thus limiting their application. Once absorbed on GO, the complex exhibited reduced toxicity and enhanced transfection efficiency compared to PEI itself.[113] Feng and coworkers[114] introduced dual-polymer-functionalized GO (NGO-PEG-PEI) via amide bonds to deliver DNA and siRNA. Their studies showed that cellular uptake of NGO-PEG-PEI was significant enhanced for the increased cell permeability even upon a low power NIR laser irradiation. Their study was the first example to illustrate light-controlled gene transfection. In a similar study, RGO, instead of GO, was used to construct a photothermal controlled gene delivery system PEG-BPEI (branched polyethylenimine)-RGO/PDNA to improve gene transfection efficiency upon NIR irradiation.[115] In another modified version of nano-carriers, a PEI (25 KDa) and PEG (10 KDa) dualfunctionalized RGO (RGPP) nanocarrier was engineered to show improved stability and higher gene delivery ability than

4.3 Phototherapy Phototherapy, utilizing light radiation to treat various disease especially malignancies, is emerging as a promising approach for cancer therapy, including photothermal therapy (PTT) and Photodynamic therapy (PDT). These treatments destroy cancer cells by either an immediate photothermal damage converted from absorbed NIR[121] or production of high concentrations of reactive oxygen species (ROS),[122] such as singlet oxygen, free radicals, peroxides anions and active cytotoxic agent within the irradiated cells. Graphenebased nanomaterials have been investigated as promising materials for PTT, owning to their suitable photothermal properties, and they can also load photosensitizers to exert influence of PDT. With regard to photothermal therapy of graphene-based nanomaterials, under irradiation with near infrared ray, the photon energy is converted to heat via non-radiative decay transitions, thus the cancer cells are killed by the continuous hyper-

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Figure 7. (a) The application of PPa-nGO-mAb. (b) The synthesis of PPanGO-mAb and its two different status. Reproduced from ref [131]. Copyright (2015) ACS Appl Mater Interfaces. (c) Cell viabilities of B16F0 cells internalized with GO-PEG-folate and treated in the dark and under photo irradiation conditions (808 and 980 nm) at 37℃. (d) The ROS levels monitored by DCF fluorescence using flow cytometry for B16F0 cells internalized with GO-PEG-folate, followed by photo irradiation with NaN3 pretreatment at 37℃. Reproduced from ref [36]. Copyright (2016) Biomaterials.

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thermia. Application of naonomaterials, like goldnanorods,[123] silver nanoparticles,[124] carbon nanotubes[125, 126] have expanded rapidly in biomedicine research, ascribed to their large surface to volume ratio and high absorption in NIR. However, metal nanomaterials are not stable or even changed structurally under NIR. In contrast, graphene and GO show dramatically improved stability after long-term exposure to NIR, thus making them ideal for PTT.[64] How to improve the performance of PTT agents. Photothermal conversion ability (PCA) and cell internalization ability (CIA) are two key factors. To improve the PCA, Su et al.[127] put forward a porphyrin immobilized nanographene oxide (PNG) grafted with tripeptide L-arginyl-glycylLaspartic (RGD), which selectively targeted brain cancer cells with enhanced therapy efficacy and no obvious side effects were observed for such materials. In vitro study indicated that 78.07% ± 0.1% of U87-MG cells were killed after 10 minutes NIR. Notably, significantly suppressed tumor growth was observed in vivo studies without tumor recrudescence while the same dosage of GO did.[128] Gao et al. developed a fluorescent/photoacoustic imaging guided PTT agent CPGA by conjugating GO/Au complex (GA) with Near infrared dye (Cy5.5) labeled-matrix metalloproteinase-14 (MMP-14) substrate (CP) and their study demonstrated elevated photothermal effect compared to GO or Au alone. Recently, Li et al. reported folic acid (FA)-functionalized graphene quantum dots (GQDs-FA), which possessed exceptionally high loading capacity (33.19%) and improved solubility by over 2400-fold for IR780. Under 808 nm laser irradiation, IR780/GQDs-FA could achieve complete tumor disappearance without relapse.[129] Previous studies mostly focus on improving the PCA, little attention has been attached on CIA. In order to further improve the performance of PPT, Du et al. investigated the gradually enhanced phototherapy effect by simultaneously changing the PCA and CIA of photothermal therapy (PTT) agent. They synthesized the hybrids of graphene and gold nanostar (GGN) and the reduced rGGN grafted by bovine serum albumin (BSA) or BSA-FA (folic acid). Compared with GGNB, rGGNB showed enhanced PCA. Besides, with the aid of the 1,2-dioleoyl-3-trimethylammoniumpropan (DOTAP) which can activate the endocytosis and promote the CIA of rGGNB, rGGNB-FA could further internalized into the cells, achieving the cancer cells death even at the low laser density of 0.3 W cm−2.[130]

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PDT has been approved by FDA for local treatment of certain cancers[132] by combining light, a photosensitizer (PS), and molecular oxygen. Targeted delivery boost quickly with reduced side effect and dose of PS. At present, the poor tissue penetration for this strategy is typically a limiting factor. To tackle this problem, Sun and coworkers[133] reported an ultrasmall and low-toxicity graphenesponge capped with lipids and lactoferrin (Lf) to deliver both hydrophobic DTX and PFH (energy) to serve as an effective penetrating and photothermal agent in the NIR region. The constructed Lf-liporGO@PS/SiO2/PFD/DTX, combining tumor penetration and NIR-guided energy/drug release, showed considerable tumor suppression. Delivery of PS to tumor site may not be sufficient to elicit maximum therapeutic response. To achieve better therapeutic effect, dual-targeting (cellular targeting and subcellular targeting) nanosystem has been designed to reach the intracellular target sites. Xu et al.[134] successfully fabricated a dual-targeting PDT nanosystem (FA-PEG-NGO/MitoTPP) to target folate receptor (FR) over-expressed cancer cells and subsequently the mitochondria to exert PDT action. This nanohybrides exhibited significantly enhanced cytotoxicity towards target cells. In another report,[131] the integrin αvβ3 monoclonal antibody (mAb) and phototoxicity agent Pyropheophorbide-a (PPa) dual-decorated nanodrug (PPa-NGO-mAb) with phototoxicity on/off switching was designed (Fig 7a,b). This nano-system achieved tumor and mitochondria two-step-

ACCEPTED MANUSCRIPT cin and Irinotecan-loaded GO; GO-DI) (note: the scales used in this reaction schematic do not denote the real situation). Reproduced from ref[140].

targeting and significantly enhanced mitochondria-mediated apoptosis of PDT.

4.4 Combination therapy

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In a recent study, Zhang et al.[141] proposed octreotide (OCT) decorated NGO-PEG-OCT nanocomplex for the first time to achieve somatostatin receptor-mediated tumor-specific targeted delivery, offering a remarkable tumor-specific uptake, cytotoxicity, and decreased side effects, and further enhanced effects by combined chemotherapy and PTT. Swain and coworkers reported a polymer stabilized iron oxide-graphene (PIG) with excellent stability and biocompatibility.[142] Data indicated PIG could load and release hydrophobic and hydrophilic drugs with high loading efficiency to realize a synergetic effect by both drug and hyperthermia. In another study,[143] ultrasmall plasmonic gold nanorod vesicle (RGO-AuNRVeDOX) showed low-power NIR laser-induced photothermal properties and a dual photo- and acid-induced drug release. Additionally, the small size (65nm) allowed perfect passive accumulation in tumor, ultimately leading to a maximized tumor inhibition and minimized side effects. Recently, Yao et al. devised graphene quantum dots-capped magnetic mesoporous silica nanoparticles (MMSN/GQDs) as a multifunctional platform, realizing controlled drug delivery, magnetic hyperthermia and photothermal therapy. The MMSN/GQDs nanoparticle could generate heat to the hyperthermia temperature by near infrared irradiation or under an alternating magnetic field, resulting in a higher efficiency to kill cancer cells.[144] Tran and co-workers developed a dual-in-dual synergistic therapy based on dual drugs (doxorubicin and irinotecan)loaded GO (GO-DI) stabilized with poloxamer 188, and combined with PTT to overcome drug resistance (Fig 8b). [140] The combination of doxorubicin (DOX) and irinotecan (IRI) could induce synergistic therapeutic effect by regulating the synthesis of DNA topology/nucleic acid in the cell nucleus (Fig 7b). This GO-DI system induced tumor cell apoptosis by up-regulation of p53, p27, and p21, showing great potential for cancer therapy and overcoming drug resistance.

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Combination therapy has emerged as a more effective strategy by the collaboration of two or even more forms of treatments. Due to space limit and availability of extensive literature on this topic, this section is focused on drug and gene codelivery strategy, photo-chemotherapy strategy, photo-gene therapy strategy and combined PTT and PDT strategy, as listed in table 1. Drug and gene co-delivery strategy has shown enhanced inhibition on tumor growth compared to drug or gene alone. Nanoparticles of poly (amidoamine) dendrimer-grafted gadolinium functionalized NGO (Gd-NGO-PAMAM) has been developed to effectively deliver both epirubicin (EPI) and Let7gmiRNA to cancer cells. Considerably high transfection efficiency and enhanced inhibition on cancer cell growth were observed.[135] Results from MR imaging also echoed this notion. A similar study conducted by Gu et al. introduced a polyamidoamne dendrimer functionalized graphene oxide (GO-PAMAM), which realized loading doxorubicin (DOX) and MMP-9 shRNA plasmid at the same time for breast cancer therapy. [136] RGO has been extremely studied in photo-chemotherapy strategy. To functionalize RGO, both reducing functionalized GO and modifying RGO directly will work. Hu and coworkers[137] employed the octadecanoic acid (C18) as a hydrophobic anchor to modify RGO directly via non-covalent bond, owing to the lack of functional groups on the edge of RGO after chemical reduction. The established FA-Dextran-C18RGO/DOX exhibited high cytotoxicity upon NIR irradiation. Furthermore, their future study [138] put forward a facile strategy to produce dextran grafted RGO in one pot, using dextran as a reducing agent. RGO–PEG/PEI was recently developed in a similar method by simultaneously reducing PEGylated nanographene oxide (NGO–PEG) and connecting RGO-PEG with polyethylenimine (PEI 1.8 kDa) in one step, as shown in Fig 8a. The constructed nano-system realized ~20-fold increment in NIR absorption at 808 nm and ~2.7-fold increment in doxorubicin (DOX) loading than GO, and ultimately achieved remarkable anticancer effects.[139]

Table 1. Functionalized graphene applied in combined therapy. Type of application

Graphene

Type of cancer

Type of tumor cells

Refererence

Drug and gene cotherapy

Gd-NGOPAMAM

Malignant brain tumors

Human glioblastoma (U87) cells

[135]

Drug and gene cotherapy

GO-PAMAM

Breast cancer

MCF-7 cells

[136]

Chemotherapy and PTT

FA-DextranC18-RGO

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Hela cells

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B16F10 cells

[138]

RGO–

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4T1 cells

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Chemotherapy and Figure 8. (a) Schematic illustration of the synthetic procedure of nRGOPTT PEG/PEI/DOX. Reproduced from ref [139]. (b) Schematic diagram showing the antitumor activity of dual drug-loaded graphene oxide (Doxorubi-

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ACCEPTED MANUSCRIPT singlet oxygen and heat upon activation by NIR to inhibit tumors, exerting nanomaterial-mediated PDT and PTT therapy without co-presence of any photosensitizers (Fig 7c,d). [36]

PEG/PEI

Chemotherapy and PTT

NGO-PEGOCT

Breast cancer

MCF-7 cells

Chemotherapy and PTT

PIG

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Hela cells

[142]

Chemotherapy and PTT

RGOAuNRVeDOX

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U87MG cells

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Chemotherapy and PTT

MMSN/GQDs

Breast cancer

4T1 cells

[144]

[140]

4.5 Tissue engineering

GO-C60

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Hela cells

[145]

PTT and PDT

NGO-PEGBPEI

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A549, Lewis

[146]

PTT and PDT

GO-PEGfolate

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B16F0 cells

[36]

PTT and PDT

GO-FA/Py-γCD/C60

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Hela cells

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dual gene co-therapy and PTT

FA-PEG-GOFAH

pancreatic cancer

MIA PaCa-2 cells

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The integration of PDT and PTT has aroused vast attention for the capacity to exceed the individual therapeutic effect. To name a few examples, Li et al.[145] introduced a new strategy to construct a GO-C60 hybrid modified by methoxypolyethylene glycol (mPEG). GO passed light energy to C60 to generate large amount of ROS upon NIR excitation, consequently this hybrid exhibited superior inhibition on tumor growth that had not been observed individually. For another successful example,[146] IR 808, a PDT photosensitizer with both NIR sensitivity and cancer targeting ability, was conjugated to both polyethylene glycol (PEG) and branched polyethylenimine (BPEI) grafted NGO (NGO-PEG-BPEI) for both PTT and PDT. Under irradiation of 808 nm wavelength, NGO and IR 808 worked simultaneously to initiate responses for PTT and PDT, ultimately leading to generate local hyperthermia and significantly high level of ROS to kill tumors with maximal phototherapeutic effects and minimal side effects. Hu and coworkers fabricated GO-FA/Py-γ-CD/C60 nanohybrid via host–guest chemistry. Folic acid grafted-graphene (GO-FA) and pyrenebutyric acid modified γ -cyclodextrin (Py-γ-CD) were linked via covalent bond to host pristine C60 molecules. Results indicated that the nanohybrid hindered the aggregation of C60, promoted the light absorption for simultaneous PDT and PTT, and thereby killed more cancer cells, compared with individual γ-CD/C60 (PDT) or GO-FA (PTT).[147] Recently, Kalluru et al. proposed that GO-PEG-folate could generate

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Tissue engineering, repairing and regenerating damaged tissues and organs caused by disease, trauma, age or congenital defects, is a rapid emerging interdisciplinary field.[149] Tissue engineering (TE) and regenerative medicine (RM) represent areas of increasing interest, with the major progress in cell and organ transplantation, as well as advances in materials science and engineering.[150] To date, various natural and synthetic biopolymers have been employed to fabricate biomimetic and fully functional tissue substitutes. Nevertheless, it is generally not satisfactory with some unreasonable properties, such as poor electrical conductivity, low mechanical strength, etc. In order to improve the performance, some other polymers (polycaprolactone (PCL), polyacrylamide, polyvinyl alcohol (PVA), etc) and conductive nanomaterials (polypyrrole, silver, gold, carbon nanotubes (CNTs), etc) are incorporated. In recent years, the outstanding mechanical strength, stiffness, electrical conductivity, various two-dimensional (2D) and three-dimensional (3D) morphologies, capability of in vivo degradation and excretion make graphene-based materials good candidates for tissue engineering, including wound healing, stem cell engineering, regenerative medicine, cell growth and differentiation, etc. It can be used as a reinforcement material to construct hydrogels, films, fibers, foams and other tissue engineering scaffolds. By now, graphene-based nanomaterials have been widely explored in bone, neural, cardiac, cartilage, musculoskeletal, and skin/adipose tissue engineering.[151] As carriers, graphene-based nanomaterials have a dose-dependent toxicity. However, when it is used as a reinforcement material to construct hydrogels, films, fibers, foams and other tissue engineering scaffolds, it does not kill cells. Instead, it accelerates the proliferation and differentiation of cells. The specific mechanism is still a mystery. Maybe when acting as carriers, these nanomaterials must be well dispersed in the blood. Their accumulation and interaction with proteins will easily lead to immune responses and even the death of cells. However, once these materials are prepared to films, hydrogels, fibers and other 2D or 3D scaffolds, cells will adhere to their surfaces for further proliferation or even differentiation, utilizing their specific surface properties. It is well known that more hydrophilic surface is beneficial for cell attachment, spreading and proliferation. Hence, GO and RGO are more suitable for tissue engineering. GO have more hydrophilic surfaces for manufacture than RGO, while the more defects on surface disrupt the electronic structure. Taken together, maybe functionalized RGO is one of the most approaches. Successful bone tissue engineering requires at least three elements: osteoprogenitors, growth factors, and scaffolds. Graphene and graphene oxide, as a multiple platform, could interact with various chemical inducer to accelerate stem cell differentiation.[152] Díez-Pascual et al. presented PPF (Poly (propylene fumarate))/PEG-GO nanocomposites with great potential to be applied in bone tissue engineering. Along with the increase of GO concentration in the composites, surface roughness, hydrophilicity, water uptake, protein absorption

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(PVA) and graphene nanosheets. 1 wt% Gr improved the toughness of hybrid scaffold due to the strong interaction between graphene and AP matrix, further to stimulate the electrical conductivity of pure AP scaffolds. MTT assay revealed that the Gr-AP scaffolds obviously accelerated the attachment, spreading and proliferation of PC12 cells for 7 days culture (1.4 fold than pure AP).[162] Topographical cues of culture substrate regulate the behavior of stem cells. Yang et al. developed a GO-based hierarchical patterned substrate (GPS) with ability to stimulate adhesion and differentiation of neuronal cells and improve electrophysiological functionality of differentiated hNSCs like sodium currents and action potential, exhibiting potential for treating neurodegenerative diseases and neuronal disorders.[163] More interestingly, a selfpowered electrical simulation system for stem cell neural differentiation was designed by Guo and coworkers (Fig 9). The human-motion-driven self-powered triboelectric nanogenerator (TENG) provided by electrical stimulation signals through the RGO−PEDOT (poly(3,4-ethylenedioxythiophene)) hybrid microfibers could induce MSC differentiation into neural cells with greater neural-specific proteins and more gene expressions.[20]

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capability, thermal stability and biodegradation rate were found to rise gradually, which was beneficial for cell adhesion and proliferation.[153] Kumar et al. prepared a macroporous 3D scaffolds (PCL/RGO_Sr) by incorporating strontium metallic decorated RGO hybrid nanoparticles into poly (εcaprolactone) (PCL), which could elute strontium ions in an aqueous medium. Osteoblast proliferation and differentiation in PCL/RGO_Sr scaffolds was apparently higher than that in PCL and PCL/RGO group.[154] Lyu et al. designed a selfsupporting graphene hydrogel (SGH) film, which had higher mechanical strength and flexibility, to induce the osteogenic differentiation of human adipose-derived stem cells (hADSCs). Interestingly, they found SGH could stimulate osteogenic differentiation of hADSCs in the absence of chemical inducers but the effect was weaker than that induced by the osteogenic.[155] In another report, a 3D porous RGO composite, prepared from nano-hydroxyapatite (nHA) and graphene oxide (GO) via self-assembly without any reducer for bone tissue repair, was introduced by Nie et al. In vitro studies manifested that this scaffold stimulated proliferation, alkaline phosphatase activity (ALP) and osteogenic gene expression of rat bone mesenchymal stem cells (rBMSCs). In vivo studies, the circular calvarial defects of 4 mm diameter in rabbit were healed more quickly by 20% nHA incorporated RGO (nHA@RGO) porous scaffold without obvious inflammation and necrosis after 6 weeks implanting. Moreover, once this scaffolds were implanted, the reconstruction and integration at the defect site occurred, evidenced by histological examination of the cranial bones.[21] Autogenous nerve transplantation is the preferred solution for nerve repair, while the limited donor sites and neurologic deficit have failed to be solved. Neural tissue engineering is regarded as the optimal alternate method. Neural tissue engineering is facing great challenges for the high organization and connection of neurons. In order to promote differentiation of neural stem cells into neurons, varieties of stimuli have been used, such as electrical stimulation, laser stimulation, flash-photo stimulation, NIR stimulation, chemical stimulation, morphological stimulation, etc.[156] It has been reported that the neuronal affinity could be tuned from separation to adhesion with the increasing crystalline quality of graphene.[157] Moreover, the outgrowth and branching of neuronal processes could be controlled by manipulating the charge of functionalized GO.[158] The differentiation of neural stem cells (NSC) is sensitive to electoral signals.[159] It is predicted that the enhanced internal electric field on graphene substrate might alter the membrane's bioelectric properties, therefore leading to the maturation and differentiation of neural stem cells.[160] Akhavan et al. fabricated electrically conductive 3D-scaffolds by precipitation of chemically exfoliated GO sheets in an aqueous suspension at 80 ℃ under UV irradiation. Consequently, the graphene oxide foams (GOFs) were rolled to form cross-sections with high hydrophilic nature to promote differentiation of human neural stem cells (hNSCs) into neurons throughout the pores and interfaces of the scaffold. Via electrical stimulation, more cells differentiated into neurons (rather than glia).[161] Golafshan and coworkers employed electrospinning technique to fabricate hybrid Gr-AP scaffolds, which was composed of sodium alginate (SA), polyvinyl alcohol

Figure 9. (a1~a6) SEM images of rGO or rGO-PEDOT microfibers made from 8mg/mL GO suspension containing 0, 1, 5, 10, 15 and 20 vol% of 50μg/μL PEDOT solution, respectively. The induced voltage (c1), current (c2), transferred charge (c3) driven by walking steps, and the stability of current output (c4). The two-dimensional structures of fluorescence morphologies of actin cytoskeleton of the MSCs cultured on rGO (d) or 15% rGO-PEDOT (f), and their three-dimensional structures (e), (g), respectively.

Cardiac tissue injury is one of the fatal diseases and heart transplantation is the only choice to cure large-scale cardiac injuries. Recently, more attentions are attached to engineer cardiac tissue to replace damaged tissues. The better strategy to construct biomimetic scaffolds is to use native extracellular matrix (ECM) components, such as collagen, fibrin, or laminin. However the poor mechanical properties and electrical conductivity hamper their further application. Shin et al. incorporated RGO with gelatin methacryloyl (GelMA), a modified denatured form of collagen, to fabricate a hybrid hydrogel scaffold. The synthesized hydrogel scaffold showed obviously enhanced mechanical properties and electrical conductivity, and promoted cell viability, proliferation, and maturation.

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(MRI), ultrasonography (US), optical imaging and multimodality imaging, allowing to detect and characterize disease in the early stage and intuitively monitor the treatment response in vivo. Besides, according to their inherent pH sensitivity and the different pH values between normal cells and tumor cells, a pH-responsive therapeutics release could be achieved, beneficial for cancer therapy. In addition, due to the distinct optical properties, these materials can not only be directly applied for phototherapy, but also achieve rapid release of the cargos from the surface via near infrared ray auxiliary irradiation as well as light controlled endocytosis by enhanced cell permeability. Furthermore, other extra stimuli, including magnetic fields, heat and energy, are also employed for accurate treatment. Recently, more innovative modalities are proposed to driven the development of these materials, like design strategy of dual-targeting and more complex combination therapy. Interestingly, the outstanding mechanical strength, stiffness, electrical conductivity, various two-dimensional (2D) and three-dimensional (3D) morphologies could stimulate proliferation and differentiation of stem cells to specific lineages, including bone, neural, cardiac, cartilage, musculoskeletal, and skin/adipose, making them promising candidates for tissue engineering. Besides, their antibacterial activity can be applied in wound sites to prevent infections. Though most are still in primary stage, great encouraging advances have been seen. However, some challenges should be overcome against time. The underlying mechanisms and signal pathway are still not clear. The potential nanotoxicity when applied in tissue engineering and regeneration medicine requires further in-depth investigations. The advancements have driven these nanomaterials to clinical applications. Along with the deep insight to them, more problems appeared and one of the concerns for further development is the potential nanotoxicity. Unfortunately, the experimental results on toxicology studies of these nanomaterials are still not conclusive to date. Because, so far, no standard production process is available, resulting in the difference of size, thickness, oxidation degree, functional proportion, dispersion degree, stability or other properties of obtained graphene-based nanomaterials. The differences influence the interaction between these nanomaterials and experimental models, thus leading to conflicted results. In addition, the different experimental models and methods are also responsible for this controversy. While the currently available results all support further development of these nanomaterials in clinical studies. With small size, reasonable surface functionalization, excellent biocompatibility, these nanomaterials appear to be less harmful. In conclusion, it can be foreseen that all issues will be solved with the continuing efforts in this regard and explosive interests to the graphene-based nanomaterials. For the future perspectives, these issues should be focused, including Ⅰ) standard production process; Ⅱ) reasonable evaluation criteria of the relative toxicity; Ⅲ) the long-term toxicity and environmental hazards; Ⅳ) underlying degradation and excretion mechanism; Ⅴ) integral understanding of the interaction between these nanomaterials and biological system. We envision

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Moreover, Cardiomyocytes showed stronger contractility and faster spontaneous beating rate on rGO-GelMA hydrogel sheets compared to those on pristine GelMA hydrogels and GO-GelMA hydrogel sheets.[164] Given the repair capacity of articular cartilage, it is of critical importance to construct tissue engineering for joint disease and trauma. Cao et al. fabricated Chitosan/Poly (vinyl alcohol) (PVA)/graphene oxide (GO) composite nanofibers via electrospinning technique. Compared with chitosan/PVA/GO (4 wt%) and chitosan/PVA, chitosan/PVA/GO (6 wt%) was most suitable for the proliferation of ATDC5 cells.[165] Liao et al. fabricated a novel CSMA/PECA/GO hybrid scaffold with adequate pore size, porosity, compression modulus, swelling ability and conductivity for cartilage tissue engineering. This scaffold was manufactured with biocompatibility and degradability. Cartilage cells seeded on the hybrid scaffold to retain chondrogenic property, which in turn achieved a better remodeling of regenerated cartilage.[166] Some other applications on tissue engineering also develop rapidly. Mesenchymal stem cells (MSCs) are promising candidates for musculoskeletal tissue engineering, which could differentiate into specific tissues, including bone, muscle, and cartilage. Graphene oxide (GO)-doped poly (lactic-co-glycolic acid) (PLGA) nanofiber scaffolds were fabricated by Luo et al. via electrospinning technique. GO not only enhanced the hydrophilic and absorption of protein and inducer, but also accelerated the adhesion, proliferation and differentiation towards osteoblast of human MSCs (hMSCs).[167] Poor engraftment, deficient vascularization, and excessive scar formation limit the regeneration of skin. In order to improve skin wound healing, Li et al. designed graphene foam (GF) scaffold to promote the proliferation of bone marrow derived mesenchymal stem cells (MSCs). They found that the regenerative dermis was thicker and more complex after two weeks transplantation. Neo-vascularization was also observed, related with the increase of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Moreover, reduced scarring was realized by a down-regulation of transforming growth factor-beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) together with an up-regulation of TGF-β3.[168]

5. Conclusion and future perspectives

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The advancements of graphene-based nanomaterials have been pushed to an exciting climax in the field of biomedicine for their diverse properties. The large surface endows graphene with the ability to absorb or bind more therapeutics to detect and treat various diseases, especially cancers. Functionalization via physical interaction or chemical reaction with the abundant active groups could achieve improved biocompatibility, reduced toxicity and special performance, like targeting. Drug, gene, photosensitizer, fluorescent dye and other molecules could reach target successfully by constructing delivery system with enhanced therapeutic effects and decreased side effects. Graphene-based nanomaterials provide a platform for various imaging techniques, including radionuclide-based imaging (PET and SPECT), photoacoustic imaging (PAI), computed tomography (CT), magnetic resonance imaging

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GO, with rich oxidative groups, such as epoxy, hydroxyl, and carboxyl groups on its surface, is obtained by highly chemical oxidation and exfoliation route upon nature graphite. GOCOOH owes more carboxyl groups on the surface by converting hydroxyl of GO to carboxyl groups via reacting with chloroacetic acid in alkaline conditions. RGO is obtained by further reduced of GO or GO-COOH through chemical reduction, thermal treatment, or irradiation, which resembles the structure and properties of graphene.

Corresponding Author

Notes The authors declare no competing financial interest.

Acknowledgements

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This work was supported by The Fundamental Research Funds of Shandong University (2016JC025).

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*E-mail: [email protected]; [email protected].

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Abbreviates

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that graphene-based nano-systems represent an innovative mean to facilitate the development of novel treatment agents, ultimately benefit pharmacotherapy development for human diseases, especially for human cancers.

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Graphical abstract

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The pharmacokinetics (including absorption, distribution, metabolism and excretion) are summarized using mice or rats as animal models, and the in vivo toxicity of graphenebased nanomaterials to zebrafish, mice, rats, rabbits and canines is discussed .

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The advanced applications of graphene-based nanomaterials in biomedicine, including bioimaging, drug/gene delivery, phototherapy, multimodality therapy and tissue engineering, are comprehensively elaborated and discussed.

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Preclinical biocompatibility of graphenebased nanomaterials in vitro is discussed, including toxicity to prokaryotic cells and eukaryotic cells, hemocompatibility, as well as inflammatory responses.

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Properties of graphene-based nanomaterials are summarized and explained in detail.

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Highlights