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Cite this article: Akhina H, Aswathy MK, Ajitha AR, Thomas S (2018) Graphene Based Materials for Human Welfare. JSM Nanotechnol Nanomed 6(2): 1067.

*Corresponding author

Akhina H, International and Inter University Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, Kerala, India, 686560, Email:

Submitted: 02 June 2018Accepted: 30 June 2018Published: 30 June 2018

ISSN: 2334-1815

Copyright© 2018 Akhina et al.

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Keywords•Graphene•Human welfare•Drug delivery carrier

Research Article

Graphene Based Materials for Human WelfareAkhina H*, Aswathy MK, Ajitha AR, and Sabu ThomasInternational and Inter-University Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, India

Abstract

Since the discovery of graphene, studies on its biocompatibility has been conducted by several researchers. Of course, its emergence turns light to tremendous application potential including biomedical applications. In this article, we included some of the facts about the biocompatibility of graphene and graphene based materials and its use as sensor for cancer diagnosing and as anticancer drug delivery carrier.

INTRODUCTIONThe development of new and effective materials with

ambient potential for saving human life is one the major issues in the biological field. With the advancement of nanoscience and nanotechnology a number of nanomaterials like nanoparticles, nanotubes, nanowires, and nanorods have been emerged. These nanoparticles can be used to carry drugs and imaging agents, which are loaded on or within the nanocarriers by chemical conjugation or simply by encapsulation.

Recently, graphene has been proved as a promising candidate for biomedical applications [1]. Graphene is a two dimensional sheet of sp2 hybridized carbon atoms densely packed into a honeycomb lattice. The structure of graphene and graphene oxide are shown in Figure 1. It has excellent physical and chemical properties owing to its structure. Graphene possesses remarkable physical–chemical properties, including a high Young’s modulus, high fracture strength, excellent electrical and thermal conductivity, fast mobility of charge carriers, large specific surface area and biocompatibility [2].

The planar π-conjugated structure of Graphene offers an excellent capability to immobilize a large number of substances, including metals, drugs, biomolecules, fluorescent probes and cells. Therefore, it is not surprising that graphene has generated great interest in nano medicine and biomedical applications, where suitably modified graphene can serve as an excellent drug delivery platform for anti-cancer/gene delivery [3], biosensing [4], bioimaging [5], antibacterial applications [6], cancer therapy and tissue engineering [7].

Now a day cancer is considered as one of the leading causes of health loss and death. The conventional techniques for cancer detection such as centrifugation, chromatography, and fluorescence and magnetic-activated cell sorting rely on the expertise and subjective judgment of highly skilled personnel. Diagnosing a cancer via cross sectional imaging (CT scan) and biopsy is an expensive and often uncomfortable approach for patients and yield substantial false-negative rates and a limited potential for early diagnosis of disease [8]. However its earlier detection and effective treatment may help to save the human life. In this chapter, we discuss the biocompatibility of graphene and its role in cancer detection and therapy.

Figure 1 Graphene (a) and Graphene oxide.

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BIOCOMPATIBILITY OF GRAPHENEIn order to use graphene and GO in real world clinical

applications, it is essential to confirm their biocompatibility and toxicity through extensive in vitro and in vivo studies using cells and animal models. However existing studies regarding the biocompatibility of graphene based materials are often contradictory. So the issues of biocompatibility have deserved great relevance. It has been noticed from literature that method of synthesis of graphene is more important in the case of biocompatibility. For examples, contrary to carbon nanotube, since metal catalysis is not used in the synthesis of graphene based materials, then cytotoxicity and inflammation caused by residual metals can be reduced [9].

It is proved that graphene based materials generally hold acceptable biocompatibility, with caution to certain factors. The effect of graphene based materials on bacteria structure, metabolism and viability has been shown to depend on the materials’ concentration, time of exposure, and physical–chemical properties, as well as on the characteristics of bacteria used in the tests. Viability was observed to decrease with increase of contact time and graphene based materials’s concentration and it has been considered that the viability decrease is associated with graphene based materials causing physical damage on bacterial membranes upon direct contact, resulting in the release of intracellular contents [6,10,11].

Kan Wang et al., studied the biocompatibility of graphene oxide on human fibroblast cells and mice. They cultured the human fibroblast cells with different doses of graphene oxides for 5 days and came into conclusion that graphene oxides exhibit dose-dependent toxicity to cells and animals, such as inducing cell apoptosis and lung granuloma formation, and cannot be cleaned by kidney [12].

In vivo toxicity of GO was studied as well, employing diverse animal models. Guo et al., reported chronic toxicity with lung inflammatory response following high intravenous dose of GO (0.4 mg) to Kunming mice [13]. GO sheets was histopathological proven to be accumulated in liver and lungs, and excreted with bile. n spite of existing disputes concerning GO toxicity, it is referred to as limited and dose dependent toxicity, with possible positive bioactivity [14]. Additionally, GO has surpassed these obstacles and attained more safety via functionalization and purification methods. Incorporation of GO using either covalent or noncovalent conjugation demonstrated alteration of surface topography and enhancement of biocompatibility [9,13].

Pristine graphene is reported to exhibit good safety profile at low concentration but own ability to reduce cell viability due to its mechanical effects and hydrophobic nature that helps in aggregation and trapping inside tissues. The mechanism behind the antibacterial activity of graphene based materials is similar to SWCNTs which involves direct interaction with the bacteria leading to irreversible membrane damage [15]. In another study, reported an increase of E. coli proliferation after exposure to GO. The GO used was dialyzed during 7 days after being produced by modified Hummer´s method. Increase in proliferation was attributed to GO acting as a support for bacterial growth [16].

Hydrophilic and small graphene based materials usually penetrate the cell membrane [12,17-19]. After internalization, these materials can lead to cytoplasmatic, perinuclear or nuclear accumulation [12,17,18]. On the other hand, hydrophobic materials are hindered from penetrating into the cell membrane due to repulsive interactions or to presenting large hydrodynamic diameters, since they tend to agglomerate in physiological medium. Despite not entering the cell, hydrophobic graphene based have been reported to cause membrane deformation, destabilize the cytoskeleton, and trigger intracellular stress, which may lead to apoptosis [17]. Hydrophilic materials may originate similar effects [12], but no relevant toxicity was observed in some cases, even after internalization [20]. It has been shown that both hydrophobic [21] and hydrophilic [5] graphene based materials generate reactive oxygen species (ROS). This is one of the major factors leading to lipid peroxidation, DNA damage, and caspase activation followed by chromatin condensation which eventually leads to cell death via apoptosis.

Thus the currently available studies on the effect of graphene based materials mammalian cell structure, metabolism and viability reveal that viability decreases inferior to 20 % after exposure to GBMs concentrations around 10 μg mL-1 during 24 h or longer [5,18,21]. The increase in contact time [12,18,20]. Also leads to a decrease in cell viability.

Nutrient depletion can be a factor contributing to the toxicity of carbon based nanomaterials. Because of its aggregation, the toxic responses of graphene sheets on adherent cell are mainly due to the limitation of nutrient availability to the cells and by generation of reactive oxygen species [21]. It must be noted that purification of the materials used is essential to assure removal of toxic contaminants.

From most of the biocompatibility studies done so far we can come in to the conclusion that the toxicity of graphene and GO is closely associated with their surface functionalization. The functionalized graphene, GO and their derivatives are shown in most cases to be significantly less toxic compared with their non-functionalized one. Considering that animal experiments conducted on rodent models could have different results from primates and humans, it is important to conduct relevant long-term toxicity studies of graphene-based nanomaterials. Hence, many more pre-clinical toxicity studies are necessary before drug delivery systems based on graphene and GO nanocarriers can be translated into clinical applications. Even though more efforts are required to further evaluate the long-term toxicology of these materials, preliminary results from the current toxicity studies provide an optimistic outlook for their successful use in biomedical applications [22].

GRAPHENE-BASED BIOSENSOR FOR EARLY CANCER DIAGNOSIS

Cancer is one of the leading causes of death in the world today. Early detection and monitoring of the advancement of cancer is very important. Biomarkers, chemical substances, found in blood urine or body tissues, play vital role in early detection of cancer. Biomarkers indicate changes in the expression of a protein that is correlated to risk or progression of a disease or its response to treatment, and that can be measured in tissues or in the blood.

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For high risk patients, such as those with a genetic pre-disposition to cancer and/or those who have already been diagnosed and treated for the disease, periodic monitoring for early detection of recurring cancer has been shown to be vital to long term survival rates in these types of patients. Currently, these patients must undergo continuous examinations and occasional biopsies by their health care professional to monitor the regression or progression of the disease. These procedures are expensive, time-consuming and invasive.

Myung et al., fabricated a graphene based biosensor for selective and sensitive detection of key biomarker for breast cancer (Figure 2) [23]. The graphene oxide encapsulated silicon oxide nano particle base field effect transistor could be used to detect any important cancer markers with relative ease. Keisham et al., developed a novel strategy of invasive diagnosis of cancer. They demonstrated that the cancer cell’s glycolysis-induced hyperactivity and enhanced electronegative membrane (from sialic acid) can sensitively modify the second-order overtone of in-plane phonon vibration energies (2D) of interfaced graphene via a hole doping mechanism [24].

In another study, graphene oxide (GO) has been incorporated in paper matrix for cancer biomarker detection. Du et al., described a promising graphene platform for the detection of cancer biomarkers [25]. They developed a novel electrochemical immunosensor for sensitive detection of cancer biomarker r-fetoprotein (AFP) that uses a graphene sheet sensor platform and functionalized carbon nanospheres (CNSs) labeled with horseradish peroxidase-secondary antibodies (HRP-Ab2). Wang et al., reported that GO can be used as cancer cell surface marker, integrin ανβ3. They studied that cyclic RGD (Arginylglycylaspartic acid) peptide and integrin ανβ 3 as a ligand–receptor pair for GO-based biosensing due to the vital role of integrin in cancer cell adhesion, proliferation, migration, and metastasis. Due to

p-stacking interactions, GO can detect the surface of cells with the help of a spectrofluorimeter [23].

GRAPHENE AND GO AS DRUG NANO-CARRIERS FOR CANCER THERAPY

An ideal drug carrier will direct the therapeutic agent to their site of action and protect them from degradation, thereby reducing off-target effect and dilution and potentially increase the efficacy. This requires a stable and inert carrier whose physicochemical properties permit its transport through complex physiological environments, while specific characteristics ensure cell specific uptake and delivery. Now a day’s various nano materials having tailored properties are used as drug carriers.

Among various nanomaterials Graphene based materials have been extensively explored in the past several years as a novel drug nanocarrier for the loading of a variety therapeutics, including anti-cancer drugs, poorly soluble drugs, antibiotics, antibodies, peptides, DNA, RNA and genes [26-28] due to its processablity and exceptional properties, Selective and controlled drug release at cancer site has reduced both cytotoxicity to normal tissue and frequency of drug administration. Various triggers are used to switch on drug release including tumor acidic micro environment, temperature changes and presence of specific analyte [22].

High specific surface area, π–π stacking and electrostatic or hydrophobic interactions of graphene can be exploited to achieve high drug loading of poorly soluble drugs without compromising potency or efficiency probably due to its high aspect ratio and abundant suface chemistry [29].

Graphene based drug delivery system has been developed by Zhao et al., by grafting the biocompatible PEGylated alginate(ALG-PEG) brushes on to the graphene oxide nano particles via the disulphide bridge bond (Figure 3) [30]. They possessed high

Figure 2 Fabrication process of biomolecular sensor based on graphene-coated NPs. a) Schematic diagram of GO assembly on amine-functionalized NPs and TEM image of NPs coated with GO. b) Fabrication of a metal electrode on the oxide substrate and surface modifi cation for the assembly of GO-NP. c) Photoresist (PR) patterns on the metal electrodes. d) GO-NP assembly in the centrifuge tube. e) Removal of PR patterns and reduction of GO coated on the NP surface [23].

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doxorubicin (DOX)-loading capacity and excellent encapsulation efficiency, owing to their unique 3-D nanoscaled structure. They also had excellent stability in simulated physiological conditions and remarkable biocompatibility. Importantly, the in vitro release showed that the platform could not only prevent the leakage of the loaded DOX under physiological conditions but also detach the cytamine (Cy) modified PEGylated alginate (Cy-ALG-PEG) moieties, response to glutathione (GSH). The accelerated release of the loaded DOX was realized in the presence of an elevated GSH that simulate the acidic endosomal compartments.

Feng et al., used the difference in pH in the tumour microenvironment to change their nanoscale graphene oxide’s tendency for uptake; the flakes were loaded with DOX and conjugated with PEG and a pH responsive polymer. In neutral or basic environments, the flakes were negatively charged, but when introduced to an acidic environment their charge becomes positive – making interaction with the negative cell membrane and subsequent endocytosis far more likely (Figure 4) [3].

Liu et al., synthesized PEG–functionalized nanoscale graphene oxide (NGO) sheets loaded with a camptothecin (CPT) analog, SN38. NGO– PEG–SN38 complex exhibited good water solubility retaining high potency and efficiency of SN38. The complex also showed high cytotoxicity in HCT-116 cells and was 1000 times more potent than CPT [31]. Jing Wu et al., investigated the reversal of drug resistance in MCF-7/ADR cells using GO as the carrier for ADR. ADR–GO with high drug loading content was prepared and its pH-sensitive release behavior was evaluated in vitro. ADR–GO could enter MCF-7/ADR cells by both endocytosis and passive diffusion, thus realizing efficient intracellular drug release by avoiding the export effects of surface P-gp. The results suggested that use of GO as a carrier of chemotherapeutic drugs is favorable for the treatment of drug resistant cancers [32].

Graphene functionalization has been successfully utilized to develop stimuli-responsive nanocarriers that release drugs in the cytosol. For example, Kim et al., exploited near infrared (NIR), acidic pH and high intracellular levels of glutathione (GSH) for intracellular cytosolic delivery of DOX. DOX, an anthracycline antibiotic, has been widely used as an anti-cancer drug in cancer chemotherapy intravenous administration. Cells treated with PEG and branched polyethylenimine (BPEI)-functionalized rGO (PEG–BPEI–rGO) nanocarriers were exposed to NIR irradiation to induce endosomal disruption and subsequent DOX release. This was further enhanced by the cytosolic presence of GSH resulting into a much higher cancer cell death in PEG–BPEI–rGO/DOX complex-treated cells with NIR irradiation than those without irradiation [33]. Yang et al., showed that the DOX molecules could make a strong bond with the GO surface through p–p interactions with quainine, the hydrophobic part of DOX, and with the hydrogen bond reaction between carboxyl groups or GO and amino groups of DOX [34]. This loading of DOX on GO is achieved by a simple mixing in an aqueous solution with the aid of sonication, showing very high loading of 235 mg ml-

1. The release of DOX was observed to be more extensive in acidic and basic conditions. Depan et al., also confirmed the pH dependence of the loading and release of DOX from GO, showing the importance of hydrogen bonding between the DOX molecule and GO [35]. This behavior is important because the pH around cancer cells is slightly acidic and therefore will promote extensive drug release of DOX. Hu et al., showed that by loading of DOX with PluronicF127 it is possible to achieve the very high loading efficiency of 289% (w/w) on GO using a low DOX concentration of 0.9 mg ml-1 [36]. The in vitro studies showed that DOX–MN– GO drug-carriers exhibit much higher cytotoxicity to SK3 human breast cancer cells compared with free DOX. The results obtained

Figure 3 Drug Release of the Drug Delivery System by GSH and pH Trigger in the HepG2 Cell [30].

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by Pan et al.[27], also indicate the potential use of GO as an ideal multifunctionalized drug nanocarrier for cancer therapy.

In the case of drug delivery, an example is that the loading ratio (weight ratio of loaded drug to carriers) of graphene nanomaterials could reach 200%, which is considerably higher compared with nanoparticles and other drug delivery systems [37,38].

CONCLUSIONThe outstanding properties, such as a large aspect ratio,

excellent mechanical strength, good electrical conductivity and unique optical properties, of graphene based material, make them ideal candidates in biomedical field. We have reviewed the applications of graphene based materials as biomarker detectors and as nano carrier for various anticancer drugs. Examples of these versatile applications have been presented here. Studies shows that up to now, numerous attentions have been paid to its applications on its cancer therapy. We envision a healthy human life with the help of these nanomaterials.

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Figure 4 Schematic illustrations of the acidic extracellular environment-induced charge reverse of our fabricated NGO-PEG-DA/DOX complex, its cellular uptake and intracellular acidic environment-triggered DOX release. Compared with wild-type MCF-7 cells, drug-resistant MCF-7/ARD cells with higher expression of P-glycoprotein show a rapid drug efflux.

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