Graphene oxide — A platform towards theranostics

MSC-07455; No of Pages 15 Materials Science and Engineering C xxx (2017) xxx–xxx

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Review

Graphene oxide — A platform towards theranostics Khazima Muazim, Zakir Hussain ⁎ School of Chemical and Materials Engineering (SCME), National University of Sciences & Technology (NUST), Sector H-12, 44000 Islamabad, Pakistan

a r t i c l e

i n f o

Article history: Received 21 November 2016 Received in revised form 2 February 2017 Accepted 24 February 2017 Available online xxxx Keywords: Graphene oxide Theranostics Composites Functionalization Biomedicine Biosensing Bioimaging Drug delivery

a b s t r a c t Due to the abundance of its utilization in various applications, ranging from electronics to biomedicine, recent data witnessed manifold increase in the commercial potential of graphene oxide based biomaterials. Major contribution of such increased potential comes from the work on graphene oxide which carries unparalleled advantage over graphene itself and/or reduced graphene oxide. Few reviews have been published in previous years which have highlighted the capacity of graphene oxide in drug delivery and photothermal therapy application. But this review exclusively provides an outlook for the role of graphene oxide based magnetic composites in constituting a theranostic system through previously inaccessible options. These composites have been exploited for their use in drug delivery applications, biosensing, bioimaging and phototherapy. This review discusses the potential challenges and advantages of using graphene oxide based magnetic nanocomposites systems to explore much needed length and breadth of theranostics, not fully elaborated so far. © 2017 Elsevier B.V. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Graphene oxide composites . . . . . . . . . . . . . . . . . . 2. Graphene oxide for theranostics (therapeutics and diagnostics) . . . . . 2.1. Drug delivery applications of graphene oxide (therapeutics) . . . 2.2. Bio-sensing applications of graphene oxide (diagnostics). . . . . 2.3. Bioimaging and photothermal therapy (PTT) using graphene oxide 3. Cytotoxicity of graphene oxide based composites in theranostics . . . . 4. Challenges and future direction . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Graphene is a single sheet of an atom thickness having sp2 hybridized carbon atoms, arranged in a honeycomb like lattice [1]. In this lattice like structure, each carbon atom is attached to another carbon atom in the same plane via covalent carbon/carbon bond. While the interlayers are arranged through weak Van der Waal forces. These forces are responsible for softness of this material. Presence of aromatic ⁎ Corresponding author. E-mail addresses: [email protected] (K. Muazim), [email protected] (Z. Hussain).

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structure, free π-π electrons and reactive sites on the periphery are the reasons for its diverse usage [2,3]. Graphene exists in many forms such as graphene sheets, graphene oxide (GO) and reduced graphene oxide (rGO) [4–6]. Properties which makes GO a material of choice in the field of biomedicine especially theranostics include its biocompatibility and biodegradability [7, 8], its large surface area (2630 m2/g approximately) [9] and high aspect ratio for modifications [10], its un-matched thermal conductivity i.e. 5000 W/m/K [11], its tendency to disperse well in aqueous medium, its better colloidal stability compared to other carbon based materials [12], its capability of traversing the plasma membrane and its cost effectiveness and scalability [13].

http://dx.doi.org/10.1016/j.msec.2017.02.121 0928-4931/© 2017 Elsevier B.V. All rights reserved.

Please cite this article as: K. Muazim, Z. Hussain, Graphene oxide — A platform towards theranostics, Mater. Sci. Eng., C (2017), http://dx.doi.org/ 10.1016/j.msec.2017.02.121

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Existing challenges for successful commercial applications of GO include reproducibility of the functionalized GO layers or composites [14, 15] and limited and/or contradictory data available on in vitro and in vivo toxicity of graphene as biomaterial [16–18] However, despite all the hurdles and difficulties in capping a suitable system for biomedical usage, GO has 62.6% research weightage towards biomedical applications in contrast to its non-medical usage [19–22]. Many of these research products ranging from synthesis procedures to their realtime applications are being patented thereby signifying their potential usage in everyday life [23–28]. There are two kinds of modifications that are generally carried out to functionalize GO nano-sheets namely, covalent and non-covalent modifications [29,30]. Covalent functionalization occurs due to mutual sharing between adjoining chemical moieties whereas non-covalent modifications include electrostatic, hydrophobic, physisorption, hydrogen bonding and π-π stacking. Presence of various functional groups such as epoxide, hydroxide and carboxyl groups provide endless possibilities to tailor covalent linkages to make desired system [31,32]. Both these modification techniques have been employed with variations and both have their own downsides. For example non covalent interactions are weak therefore show instability to external environment in vitro and in vivo while covalent modifications allow less quantity of drugs (aromatic) to be uploaded since GO sheets are also occupied by the coated polymers or other functional moieties [33–36]. 1.1. Graphene oxide composites Remarkable properties of GO are mainly associated with chemical modifications & its combine effect with various entities such as polymers and magnetic nanoparticles (Fig. 1). Since GO tends to aggregate under the physiological conditions (due to the presence of salts, ions and proteins) thereby reducing the proposed effectiveness, these modifications not only help retain its effectiveness but also reduce toxicity of the other component. Most of the composites include chemical moieties that provide biocompatibility (e.g. poly ethylene glycol (PEG), poly vinyl chloride (PVC)) [37–39], thermo/stimuli responsiveness (e.g. poly (Nisopropylacrylamide) (PNIPAM) [40,41], enhance mechanical

properties (PMMA, PVC) [42–44], used for the surface coating of the biomaterials (e.g. dextran, polyamide 11) [45] and enhance colloidal stability (Sulfonic acids, Oleylamine) [46] (Fig. 2). Therefore, a lot of research highlights the use of GO in drug delivery application [35,48,49], magnetic resonance imaging (MRI) [50–52], fluorescence imaging [53], antibacterial activity [54], biosensors [55–60] and hyperthermia [61,62]. Despite of all this available literature, there is a dearth of knowledge towards GO based magnetic nanocomposites and its role in theranostics [63,64]. This review entails the compilation of studies carried out on the GO composites used in theranostics from 2010 to 2017 (Fig. 3). In contrast to previous studies, this information is sorted and lately discussed to evaluate the potential challenges and advantages of using GO based magnetic nanocomposites for theranostics. 2. Graphene oxide for theranostics (therapeutics and diagnostics) 2.1. Drug delivery applications of graphene oxide (therapeutics) Maintaining the efficacy of therapeutic drugs is a major driving force behind drug delivery research. In order to maintain the efficacy of drugs, long term sustained release of drug through blood circulation is required. SN 38 is considered as water insoluble drug and its dispersion is an issue which hampers its potency to attack colon cancer cells. Dai et al., used PEG conjugated graphene nanosheets with non-covalent adsorption of SN38 [38]. This non-covalent adsorption was driven by hydrophobic interaction and π-π stacking. Through this system, they were able to attain controlled release along with high potency i.e. IC 50 value 6 nM for Human Colon Cancer Cell line (HCT cells) in comparison to its pro-drug, a hydrophobic analogue Camptothecin (CPT-11). Further this group used PEG-NGO for the targeted delivery of Rituxan (CD 20 antibody) and Doxorubicin (DOX) (Fig. 4). This system exhibited pH dependent drug release [65]. Zhang and colleagues tested the ability of graphene nanosheets to carry multiple anticancer drugs at a time. This approach was one of its kind and significant to reduce drug resistance occurring for many cancer treatments thereby reducing their efficacy over time. They used NGO functionalized with sulfonic acid groups which was decorated later

Fig. 1. Depiction of modifications that can be made to modify GO.

Please cite this article as: K. Muazim, Z. Hussain, Graphene oxide — A platform towards theranostics, Mater. Sci. Eng., C (2017), http://dx.doi.org/ 10.1016/j.msec.2017.02.121

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Fig. 2. Schematic illustration demonstrating strategies for the use of graphene based materials for theranostics. Adapted with permission from Ref. [47].

with folate (FA) receptors through covalent binding. FA receptors enable direct uptake of drugs which were loaded to FA- NGO matrix. The loading capacity of Dox and CPT drugs was equal to their single loading. Although therapeutic efficacy was increased but cytotoxicity to Human breast cancer cell line (MCF-7) was reduced. Efficacy was increased due to its specificity to deliver drug only to cancer cells [66]. Following the pursued, Kakran et al., functionalized GO with hydrophilic and biocompatible polymer such as Tween 80, Pluronic F38, maltodextrin and the functionalized GO was further used as a nanocarrier for poorly water soluble anticancer drug, ellagic acid (EA). For the very first time EA was loaded onto functionalized GO using ππ interaction. Release kinetics and cytotoxicity of the loaded drug formulation was evaluated at various pH. This functionalized GO carrying EA was further tested to target MCF-7and human colon Adenocarcinoma cells (HT29) [67]. It was established that GO did not hamper the antioxidant activity of loaded EA. Another research group led by Yang et al. in 2011 has explored the drug carrying capacity of graphene sheets through dual target functionalization and pH sensitivity. In this research surface of GO

sheet was functionalized with targeting ligand FA receptors and super paramagnetic iron oxide nanoparticles (Fe3O4). Multiple functionalized GO was able to demonstrate targeted and pH responsive drug delivery. In this setting, GO was decorated with Fe3O4 while 3-aminopropyl triethoxysilane (APS) was use to coat Fe3O4. This coating served as mediator to attach FA to GO-Fe3O4 composite [68]. Hybrid graphene nano-sheets with chitosan showed improved solubility in acidic medium and such functionalized hybrid showed marked controlled drug release behaviour. The drugs tested in this hybrid system were Ibuprofen and 5-fluorouracil. Microscopic techniques such as SEM and AFM were used to scan the topographic features of the functionalized graphene sheets (Fig. 5). It was first report on the adsorption of aromatic moiety containing drugs tested on graphene sheets and concluded that FGOCs has better cellular penetration and hence has better chances of success in its use in drug delivery [69]. Controlled release formulation has been made using biocompatible GO and biodegradable chitosan. In this formulation, GO was loaded with Dox and lately encapsulated with FA conjugated chitosan [70]. This formulation had shown its immense potential as targeted and

Fig. 3. Tabulated illustration of theranostic based applications of GO composites.

Please cite this article as: K. Muazim, Z. Hussain, Graphene oxide — A platform towards theranostics, Mater. Sci. Eng., C (2017), http://dx.doi.org/ 10.1016/j.msec.2017.02.121

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Fig. 4. A schematic illustration of doxorubicin (DOX) loading onto NGO–PEG–Rituxan via π-stacking. Adapted with permission from Ref. [65].

control delivery nanocarrier as this formulation was sensitive to acidic environment. In another study, GO was grafted via facile amidation reaction with chitosan (CS). This CS-g-GO was tested for its drug & gene delivery potential. It was concluded that the CS-g-GO was able to carry CPT which resulted in higher toxicity towards HepG2 and HeLa cell lines than the pure drug (Fig. 6). Similarly, gene delivery potential was illustrated through the delivery of CS-g-GO with plasmid DNA in stable and complexed form into the HeLa cell lines [71]. Effect of AuNPs on drug delivery was investigated where AuNPs were grown in-situ on the GO sheets. This nanocomposite consisting of AuNP/GO showed efficient drug delivery in the Hela cell lines. Such

nanocomposite can also be exploited towards intracellular Raman Imaging [72–74]. In 2012, Kurapati used GO along with LBL microcapsules (poly(allylamine hydrochloride) (PAH) where the composite was stimuli responsive towards near infrared light. The GO-PAH microcapsule released encapsulated drug Dox in a point wise fashion upon Near Infrared Radiation(NIR)-laser ablation. This laser ablation generates local heating effect which in turn leads to the release of drug from microcapsule. In addition to excellent optical and permeable properties of GO, it has also enhanced the mechanical strength of the microcapsule thereby preventing its breakage during intracellular delivery. Following images (Fig. 7) give a clear illustration how this microcapsule release drug in point wise fashion [74]. GO has not only been used as stimuli responsive drug delivery system but also for gel based drug delivery system. Due to recent emphasis on the use of gel matrices for the delivery of drugs such as doxorubicin hydrochloride, CPT, 5-fluorouracil, paclitaxel, cisplatin and adriamycin, exploiting the strength of novel materials is of significant importance. In order to attain these gel formulations various polymers are generally used through physical and chemical cross linking in addition to the use of chemical cross linkers such as photo initiators are used. These species though contribute good mechanical strength but are deemed inappropriate towards biomedical applications. In a recent research, GO has been used to encapsulate doxorubicin hydrochloride through gel matrix. Edge of this research was that none of the polymers or chemical matrix was used except GO which was used for in situ gelation effects (Fig. 8). Doxorubicin was released in a sustained released manner. GO-Dox gel exhibited good mechanical strength and good inject ability [75]. Another aspect of the use of this GO based hydrogel is their capacity to self-heal, carry various biomolecules (DNA) or dyes [76,77]. In another remarkable work exploiting drug carrying capacity of GO, Yang et al., used the principle of multiple supramolecular assembly to

Fig. 5. Schematic illustration of synthesis of the FGOCs and the dispersion of (a) GO and (b) the FGOCs in an aqueous acetic acid solution (CH3COOH/H2O 0.2/1). Adapted with permission from Ref. [69].

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Fig. 6. Schematic of the pH-responsive association behaviour of the amphiphlic GO–CS nanocomposites. Adapted with permission from Ref. [71].

create a GO scaffold for drug deliver [78] (Fig. 9). In this approach three components were used 1) folic acid modified β cyclodextrin, a target unit 2) adamantanyl porphyrin as linker and 3) GO as carrier unit. It was observed that due to the presence of folic acid modified β cyclodextrin GO nanocarrier could recognize FA receptors on cancer cells thereby increasing the effectiveness of Dox in comparison to free Dox. In an attempt carried out by Wu et al., it was demonstrated that the effects of drug resistance in breast cancer cells can be avoided using GO loaded with Adriamycin. Adriamycin was physically loaded onto the GO sheets. This GO-ADR caused effective reversal of ADR resistance in MCF/ ADR cells with reversal index of 8.35 [79,80]. Similarly, in another study, dual usage of GO platform was exploited where [81] authors used the ability of GO to carry single stranded DNA/RNA with ease and its loading capacity towards the anti-cancer drugs through π-π interaction was investigated. Bcl-2 which is considered to be an important anti-apoptotic defence protein which leads to multiple drug resistance (MDR) [82]. This drug resistance can be avoided by knocking down the protein's expression where the role of siRNA is extremely important since knockdown of Bcl-2 expression will not only inhibit MDR but also make cancer cells sensitive towards the anti-cancer drugs. Chemically grafted GO with polyethylenimine (PEI) was used as a nanocarrier to Dox and the Bcl2 targeting siRNA. This study showed that the PEI-GO can effectively be used as a nano-carrier and for the sequential delivery of Bcl-2 targeted siRNA. This dual effect led to the significantly enhanced chemotherapeutic efficacy. In another study, GO coupled with PEG-FA

was used to carry hTERT siRNA for the intracellular delivery of siRNA [83]. In this research, GO was conjugated with PEG-FA in order to make it bio-compatible and selective. In addition to this siRNA was loaded onto the graphene sheets with the help of 1-pyrenemethylamine hydrochloride through π-π stacking. This GO-PEG-FA-PyNH2 carried HTERT siRNA led to significant silencing of mTRET expression in HeLa cell lines. This was confirmed through RT-PCR and the western blotting (Fig. 10). In another study, hybrid of PEI modified GO with oleic acid was created which was further modified by up conversion nanoparticles (UCNP) and superparamagnetic nanoparticles. This hybrid (PEI-GO) was used as nano carrier of hydrophobic nanoparticles and resulted in the transferring of hydrophobic nanoparticles from organic phase to a water-soluble phase. PEI-GO-UCNP hybrid exhibited 100% weight loading of Dox drug. This drug carrying hybrid showed higher killing potential towards cancer cells in in vitro environment (Fig. 11) complimented by the luminescence properties due to the UCNP [84]. In a recent study, Szunerits et al., demonstrated that the composite of magnetic nanoparticles coated with 2-nitrodopamine and GO could be used for the effective delivery of insulin without damaging the native state of insulin in the acidic environment. Loading capacity of GO and GO composite was extremely high for insulin where 100 ± 3% was loaded on to GO sheet while 88 ± 3% was loaded on to GO-MPdoxmatrix. Insulin loaded onto GO-MPdox nanomatrix was protected from gastric secretions and acidic environment while drug was released once exposed to basic environment (pH = 9.2) [85]. The drug delivery potential

Fig. 7. Illustration of the remote opening of GO–polymer composite capsules using NIR-laser light. Adapted with permission from Ref. [74].

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carbodiimide (EDC) and NHS. This CTX-GO was further loaded with Dox through non-covalent interactions. This CTX-GO-DOX showed significant improvement towards the treatment of gliomas (Fig. 12). Conjugated GO was not only able to deliver drug specifically to the glioma cells but has also proved to maintain sustained release [87]. In another recent study, GO was deposited inside a conducting polymer, poly pyrrole (PPy) properties (Fig. 13). These electrical properties were further used for the release of dexamethasone which was loaded on a conducting nanocomposite [88]. 2.2. Bio-sensing applications of graphene oxide (diagnostics)

Fig. 8. Photographs for the formation of the gel matrix based on 6 mg/mL GO nanosheets and 2 mg/mL DOX. Adapted with permission from Ref. [76].

and effectiveness of GO based composites can be increased using various preparation technique. One of the studies which evaluated the difference in the efficiency of GO composites synthesized using in situ and ferro-fluid techniques. In this study it was exhibited that the GO carrying iron oxide nanoparticles in the form of ferrofluid demonstrated higher toxicity towards the MCF-7 cell lines due to higher iron content, higher loading efficiency of the drug (Anastrazole) and smaller particle size [86]. Recently GO has also been conjugated with targeted peptide of chlorotoxin(CTX) through 1-ethyl-3-(3-dimethyaminopropyl)

GO has been exploited as an essential material for bio-sensing applications since electron donor & acceptor molecules are exposed at planar surface making GO an efficient candidate for long range quenching [89–91]. In a study, it was established that COOH modified GO has an intrinsic peroxidase activity which can extend its use for glucose detection. In this report, catalytic property of GO was investigated through the use of peroxidase substrate (3,3,5,5-tetramethylbenzidine (TMB). GOCOOH has high catalytic ability compared to the naturally occurring Horse reddish peroxidase (HRP) enzyme. This ability of GO-COOH has been exploited for highly selective glucose detection. The level of glucose detected was as low as 1 × 106 mol L−1 while its linear range of the detection was 1 × 106 mol L− 1 to 2 × 105 mol L− 1 [92,93]. GOCOOH was found to be not only highly selective but also has advantages such as ease of preparation, low cost and stability. Based on the similar intrinsic peroxidase activity of the GO, it has been further exploited for the immunosensing towards Prostate Specific Antigen (PSA). In this approach, magnetic beads were modified with anti-PSA antibody (Ab1) while GO was modified with Ab2 making the entities an immunocomplex, sandwiching antigen protein PSA. Once the reaction

Fig. 9. Schematic illustration of Synthesis of 1/2/DOX/GO from graphene oxide, DOX, adamantane-modified porphyrin, and folic acid modified cyclodextrin. Adapted with permission from Ref. [78].

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Fig. 10. The preparation of functionalized graphene oxide for targeted intracellular delivery of siRNA. Adapted with permission from Ref. [83].

was complete, magnetic beads were removed while the concentration of Ab2-GO was calculated due to colour change (Colorimetric), exhibiting as if it reacts with hydroquinone and H2O2. This study established evidence that GO can be used as selective and point of care tool for clinical diagnosis. Similarly, in another study, it was reported that the intrinsic peroxidase like property of GO can be enhanced once it was decorated with iron ferrites (Fe3O4) [94]. Kinetic parameters of the study supported that the catalytic activity was enhanced under the provided settings. Following table exhibits the improvement made in the peroxidase activity of the GO based ferrites composite (Fig. 14). In a continuation, another study demonstrated the use of nanocomposite prepared from rGO and cobalt ferrites which exhibited higher reaction and subsequently higher catalytic (peroxidase) activity towards the substrate (TMB). This composite was prepared by the use of PVP as reducing agent and stabilizer and showed not only higher catalytic activity but also higher stability in the presence of different solvents and at various temperatures. However, at optimum conditions, the detection limit of this nanocomposite was 0.3 μM which was higher than many other ferrites based colorimetric sensors [95]. It has also been demonstrated in number of recent reports that both Iron, platinum

[96] and cobalt [97,98] based composites with GO having the ability to mimic enzyme like activity are likely to play an important role in biotechnology (Fig. 15) and environmental detection [99]. Recently Mn ferrites have also been explored for the colorimetric detections/peroxidase like activity [100]. However, composites of GO with Mn ferities for peroxidase activity are still to be explored. Some of the fascinating applications of graphene nanocomposites reported so far include its use in DNA sensing [89,101], protein sensing, and protein assays. To explore the ability of GO as molecular probe in situ and in vivo, Wang and coworkers investigated graphene nanosheet and aptamer with carboxyfluroscein (FAM/GO-nS) where graphene nanosheet was used as sensing plate and aptamer as molecular probe [89,102]. Since aptamers have specificity and sensitivity for the target, the target in this case was a fungi toxin (Ochratoxin A) secreted by Aspergillus ochraceus and Penicillium verrucosumm. In this case Adenosine triphosphate (ATP) aptamer was used which was non-covalently bonded to GO nanosheets. It was demonstrated that GO sheets have the ability to protect aptamer/DNA molecule from enzymatic cleavage activity and also leads to fluorescence quenching. Both these properties make it a strong contender for DNA/RNA/protein cargo for gene delivery and for bio-labelling for cellular imaging respectively.

Fig. 11. (a and b) Photographs of the phase transfer of OA-coated UCNPs green from chloroform to water. The top layers in (0 and (b) is pure water, and PEI-GO aqueous solution (0.2 mg/ml), respectively; (c) Upconversionluminescenceintensity (k = 541 nm) of PEI-GO-UCNP green in aqueous phase and OA-coated UCNPs green in chloroform during the process of phase transfer. Adapted with permission from Ref. [84].

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Fig. 12. Schematic illustration of the preparation of CTX-GO/DOX. Adapted with permission from Ref. [87].

Luo et al., in 2012 reported a convenient and enhanced chemiluminescence (CL) biosensor for sequence specific DNA detection. It was previously known that GO has fluorescence quenching property and was presumed that it will have CL property too. In this study, human immunodeficiency virus (HIV) oligonucleotide sequence associated with the HMDNAzyme (PHIV) was used as a model probe. This probe (ssDNA) once mixed with GO was assumed to be adsorbed to GO surface through π-π interaction between ssDNA nucleoside/nucleotides and GO. Since HMDNAzyme stimulates CL in the presence of luminol and H2O2 [102] and the probe ssDNA assumed to hybridize with the complementary target DNA, the presence of dsDNA assumed to be released from the GO causing significant increase in CL emission. This system was further suggested to have potential for sequence specific detection not only for DNA but for other biomolecules as well (Fig. 16). Similarly, another research group has reported the photoluminscent (PL) properties of graphene quantum dots (GQDs). These GQDs has shown excitation and solvent biased PL behaviour. Once GQDs were excited from 400 to 540 nm the PL peaks were shifted from 515 to 570 nm but with a significant decreased intensity. Also PL peak shifted from 475 to 515 nm for different solvents since GQDs were sensitive towards solvent being used. These GQDs were developed by single step solvothermal method. The PL quantum yield was as high as 11.4%. Such GQDs have advantages such as being biocompatible, low cytotoxicity and no photobleaching effect [103] and show great promise for future research.

Fig. 13. Drug loading into and release from the GO/PPy nanocomposite. Schematic representation of the (a) GO/PPy-DEX nanocomposite and (b) DEX release from the GO/ PPy nanocomposite in response to electrical stimulation. Adapted with permission from Ref. [88].

Another example of using GO as peptide biosensing platform has been illustrated in a recent study [104]. In this approach, it was proposed that GO-peptide complex can be used for real time, specific and time dependent detection of cancer biomarker. This study shows that since GO was already at quenching state due to the binding of RGDPyrene by ππ stacking to that of GO, the quenching state was restored by the competitive binding of RGD with Integrin αvβ3 where Integrin was found to play important role in the development of cancer cell's adhesion and proliferation (Fig. 17). GO based sensing has enabled scientists to overcome many limitations such as distance in the designing of efficient and sensitive FRET based biosensors. One of the studies carried out lately demonstrated the use of florescence accepting property of GO while CdTe quantum dots as F1 donor entity to pave a way for the hepatocellularcarcinoma immuno-sensor [105]. GO was conjugated with capture antibody 1(Ab1) while QDs were modified with reporter protein (Ab5) where AFP acted as a bridge. Ab1/AFP/Ab5 led to the development of selfassembled complex which resulted in the quenching of QD's emission. This GO based immuno sensor bridged the distance dependent limitations in the case of traditional immuno-sensors. In addition to sensing of DNA, GO based composite has also been used for the detection of protein. In order to detect protein, protease substrate linker was attached between energy transfer donor quantum dots and the energy acceptor GO leading to a GO-quantum dot nanoprobe. In a similar attempt, extremely sensitive insulin detection was reported [106]. It was demonstrated that GO-aptamer binding events can compete with target aptamer binding events resulting in the release of aptamer from the GO surface leading to the sensing of insulin. Furthermore, it was demonstrated that this aptamer can further be amplified employing amplification technique using DNase I enzyme. This amplification strategy led to 100-fold higher sensitivity than the traditional 1:1 binding assays while LOD reported was 5 nM. Few studies show the functionalization of GO with polyoxyethylene sorbitol anhydride monolaurate (TWEEN-20) [107] where TWEEN-20 was reacted with GO through hydrogen bonds as well as through Van der Waals forces [108]. This TWEEN-20 functionalized GO was further grafted with AuNP through in-situ chemical reduction of gold salts. Such AuNP/TWEEN-20/GO nanocomposite showed good catalytic activity towards hydrazine oxidation and the reduction of 4-nitrophenol and demonstrated the promise of exploitation in bio-sensing as well as in

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Fig. 14. Comparison of the apparent Michaelis–Menten constant (Km) and maximum reaction rate (Vm). Adapted with permission from Ref. [94].

the environmental monitoring. Similarly, in another study, GO was modified with hydroxypropyl-b-cyclodextrin (HPCD) and tetraphenyl-porphyrin(TPP) as an electron transfer molecule which led to demonstrate excellent electro-catalytic activity by detecting haemoglobin reduction or oxidation at the detection limit of 5 × 10−9 M [109]. 2.3. Bioimaging and photothermal therapy (PTT) using graphene oxide In an attempt to exploit GO based composite materials for biomedical applications, dextran coated iron nanoparticles were attached to GO through amide linkage in the presence of EDC creating a GO-Fe3O4 composite (Fig. 18). The composite was found to be stable at physiological conditions with low cytotoxicity and exhibited as an effective T2 contrast agent for cellular MRI. T2 relaxivity of this composite was enhanced due to the aggregation of iron nanoparticles on the GO sheets. In this research work it was established that the MRI ability of amino dextran (AMD) and meso-2,3-dimercaptosuccinic acid (DMSA) coated Fe3O4 was much lower compared to the Fe3O4 − GO composite. The composite was found to show T2 relaxation rates (1/T2 s− 1) as 76 Fe mM− 1 s− 1 [110]. It was reported that increasing the cellular iron content will lead to better cellular uptake resulting in contrast enhancement effect for MRI applications. In another recent study, mini-emulsion and solvent evaporation technique was employed to synthesize GO decorated with Mn ferrites nanoparticles. This composite was tested as MRI contrast agent [51]

where MnFe2O4-GO was found to show T2 relaxivity (r2) as high as 256.2 (mM Fe)− 1S− 1 with 14 nm sized MnFe2O4 nanoparticles (Fig. 19). Various reports presented above and few other studies show that GO based composite have not only been exploited for the MRI but also for the photoacoustic tomography (PAT). In a similar attempt, multimodal ability of the GO composite was investigated in a study where rGO sheet was decorated with iron oxide nanoparticles whereas PEG was non-covalently attached to the rGO. This composite was found to show triple modal imaging (fluorescence, PAT and MRI) under guided imaging PTT could be performed for the 4 T1 murine breast tumour cells. This PTT was found to exhibit efficient ablation of cells at 808 nm NIR irradiation [111]. GO has been proposed to be useful towards positron emission tomography (PET) imaging as well. In this work rGO was not only labelled with Cu but also with an antibody (TRC105) which led to specified tumour targeting in 4 T1 mice tumours [112]. Exploitation of carbon-metal combinations for the sequential drug delivery and photo-thermal effect (Chemo-photothermal effect) has been investigated [113]. During this investigation, a hybrid containing RGO-AuNRve (65 nm) was designed (Fig. 20). In this case, amphiphilic AuNRs were grafted with PEG and poly(lactic-co-glycolic acid) (PLGA) and PEGylated rGO and Dox was loaded on AuNRve and rGO thereby increasing the loading capacity of the system. Dox was released sequentially at the NIR thermal heating stimulations. Additionally, through PET imaging it was observed that hybrid vesicle was accumulated

Fig. 15. Schematic Representation of Colorimetric Detection of Cancer Cells by Using Folic Acid Functionalized PtNPs/GO. Adapted with permission from Ref. [96].

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Fig. 16. Schematic representation of chemiluminescence DNA detection relying on the discrimination ability of GO towards ssDNA/dsDNA. Adapted with permission from Ref. [102].

sufficiently thereby creating photoacoustic (PA) signals in tumour region. This research has tremendous potential for its translation to the treatment of cancer as tumours become accessible through light. GO containing various functional groups and with different nanoparticles has been exploited for their use for not only PTT but also for image guided therapy (Table 1). A recent study shows the use of AuNPs grown into PLA microcapsules followed by GO layers' encapsulation for effective photothermal ablation. These microcapsules were

investigated to not only serve as effective mediator for PTT but also as contrast agents for ultrasound (US) imaging and X ray CT imaging in in-vitro as well as in-vivo thereby, proposing a path towards next generation image guided therapy [114]. In another report, rGO composite was modified with polyphenol constituents of green tea and such modified material was further used for PTT of HT29 and SW48 colon cancer cells. PTT performed with such modified system was found to be two orders of magnitude higher than carbon based materials [115]. In another

Fig. 17. Schematic illustration of RGD–pyrene interaction with GO to quench the fluorescence of pyrene, and the fluorescence recovery by introducing either integrin αvβ3 protein or integrin overexpressing cancer cells. Size is not in scale. Adapted with permission from Ref. [104].

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Fig. 18. Schematic illustration of preparation of the Fe3O4-GO composites and T2 weighted cellular MR images: (a) HeLa cells (2 × 105 cells mL−1) incubated with the Fe3O4-GO composites at different concentrations. (b) Fe3O4-GO composites (20 μg Fe mL−1) incubated with HeLa cells at different cell density which Fe3O4 NPs formed aggregations on the GO sheets, resulting in a considerable enhanced T2 relaxivity. Adapted with permission from Ref. [110].

approach, Bovine Serum Albumin (BSA) was exploited as reducing and stabilizing agent to prepare rGO which showed promise towards dual modality imaging for PA and US in addition to its potential for PTT [116]. Effect of the preparation methods for GO composites on their performance as PTT and bio-imaging agents has also been investigated and reported [10]. Furthermore, Table 1 show the effect of the nature of GO based nanocomposite and molecule loaded onto it for bio-imaging as

well as PTT. It can be concluded that in order to exploit GO based nanocomposites for mentioned applications, there exist unlimited possibilities and by selecting the components of composite system as well as type of molecule loaded onto them, one can prepare a system with defined, desired properties and potential. Therefore, it can be envisaged to witness novel systems with enhanced performance by exploiting the strength of graphene based materials in the near future.

Fig. 19. Schematic diagram illustrating: (a) formation of oleylamine modified nanosized graphene oxide sheets (GO g– OAM), followed by (b) the synthesis of water soluble MFNPs/GO nanocomposites (MGONCs) and (c) the PEGylation of MGONCs to improve the colloidal stability of the MGONCs in physiological environment. Adapted with permission from Ref. [51].

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Fig. 20. Schematic illustration of sequential DOX release triggered by (i) remote NIR laser irradiated photothermal effect and (ii) acidic environment of the cancer cell. Adapted with permission from Ref. [113].

3. Cytotoxicity of graphene oxide based composites in theranostics Graphene oxide based composites arises from the combinatorial use of GO and magnetic nanocomposite. It is important to remember that unlike any other material GO has hydrophilic groups on its basal surface which makes this material not only available for several conjugations but also makes it more hydrophilic in nature. GO based composites so far has shown unparalleled precedent to carry out its future usage in theranostics however there are certain concerns surrounding this biomaterial. Top most of these concerns is the cytotoxicity of these materials. Many studies have shown that GO based nanocarriers do not exhibit cytotoxic activity therefore can be considered as biocompatible materials. A recent study has made an effort to standardize the effect of GO based nanocarriers on cells and animal models. They have concluded that nanosized GO (100–200 nm) do not cause cytotoxicity however micro sized GO does have a notable effect on cytotoxicity of cells (Fig. 21). This effect is due to the faster sedimentation rate and aggregate formation of microsize-GO [20]. It was also reported that surface functionalization and bioconguation of GO can lead to significant change in its physicochemical properties thereby their cytotoxic behaviour. It is possible to make GO based nanocarriers biocompatible by adding polymers such as PEG [126,127]. In vivo effect of GO has been demonstrated using monolayer cultures of fibroblasts [16,128], neuronal cells [129,130] and lung endothelial cell [131].

A lot of work has been carried out to explore the cytotoxic effect of graphene through molecular stimulations as well. These studies focus on the ability of graphene oxide to distort natural orientation of biomolecules such as aptamer and proteases. However limited studies are available on in vivo data confirming the effect of graphene or its derivatives in living cells. Few of the studies have reported cellular disruption mechanism of graphene. The mechanism of cellular disruption is characterized by distortion of outer most cellular membranes as explained in detail by [21,126]. This mechanism however has been attributed to the development of green antibiotics helping scientists circumvent problems such as antibiotic resistance and environmental protection due to chemical exposure. Data on cytotoxicity of GO based carriers remains contradictory and challenging therefore most researches today accounts for biocompatibility and cytotoxic potential of graphene and its derived composites in their performed research work [132]. Cellular mechanism of GO toxicity has been elaborated previously [133]. This is why despite all the safety concerns over a limited period of time only few number of patents have been filed for the commercial usage of GO based materials for various biomedical applications [134–137]. 4. Challenges and future direction Nanotechnology is a bourgeoning field which is helping us address some very critical issues from health to climate and somehow has become an everyday practice [138]. Recent research is oriented towards

Table 1 List of graphene oxide systems for bio-imaging and/or PTT. Composite

Molecule loaded

Highlight of the research

References

GO-PEG GO-Folic Acid (FA) nano-rGO-PEG GO-IONP-Au GO rGO/HArGO GO-BaGdF5 rGO-PEG PEGylated-GO

Chlorine e6 Chlorine e6 RGD PEG Hyaluronic acid Indocyanine green (ICG) PEG 131I CySCOOH

Better cancer cell destruction than free chlorine e6 FA-GO loaded with chlorine e6 photosensitizer for drug delivery and photodynamic therapy (PDT). Highly effective due to RGD motif for photo ablation Enhanced absorption at NIR -effective PTT with dual MRI and X-Rays imaging Enhanced transdermal delivery of GO-HA for effective PTT Image guided synergistic photo thermal effect Enhanced stability for passive targeted PTT Combine nuclear image guided radiotherapy and PTT Synergistic use for the photoacoustic imaging and PTT

[117] [118] [119] [120] [121] [122] [123] [124] [125]

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Fig. 21. In vitro compatibility of GO Hemocompatibility, macrophage uptake, immune response, cytotoxicity and cellular uptake. Adapted with permission from Ref. [126].

utilization of advancement of nanotechnology, molecular biotechnology and biochemistry to bring fundamental breakthroughs in the field of nanomedicines, especially theranostic. Although, various studies show significant improvement but much has still to be done in order to bring techniques and materials to be integrated into a practical device having high sensitivity, selectivity and acceptable reproducibility [47, 139]. 1. Properties of GO such as intrinsic fluorescence, chemi-luminescence and above all acceptable biocompatibility makes it a considerable choice for the biomedical/biomaterial based research. However, this review has been compiled by primarily presenting the work related to GO based composites used for core biomedical applications, especially theranostics. Such composites not only present multiple usages but also enable to overcome various drawbacks of the individual components such as uniform dispersion of nanoparticles in the nanosystem or increased mechanical strength of the polymer used. 2. Additionally, GO based composites also provide increased blood circulation time of the drug to circumvent multiple drug resistance and uncompromised drug loading due to their lateral dimensions. 3. Graphene/GQDs are expected to provide their diverse usage but they are still in their nascent state of work in comparison to GO based magnetic nanocomposite. Lack of control over their PL properties, optical properties and low quantum yield are the areas that require vast improvement for their successful use in theranostics. 4. GQDs are associated to have lower production yield in addition there are only fewer studies exploring the multifunctionality of GQDs in theranostics. 5. Some of the limitations of GO based composites may include concerns regarding its renal clearance and biodegradation while expectations linked with GO composite based research can only be met if scientists clarify doubts regarding its reproducibility, efficacy and toxicity on the environment and on public health. 6. It is also pertinent to mention that the contradictory data on the toxicity of GO is attributed to the heterogeneous nature of its synthesis process. Therefore, it is imperative to agree to a universal nomenclature of graphene materials created in laboratories through specific

protocols that will help avoid false expectations and unnecessary safety concerns [104]. Finally, in order to develop and translate potential patient inspired engineering solutions, further work on GO based composites materials is essentially required which will solve various clinical problems in diagnostics and/or treatment areas. In order to move nanomedicine from bench to bedside, a lot of work related to materials aspect as well as pharmaceutical or clinical aspects is warranted.

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