Hence, it ignites prevalent interests in developing a new graphene/graphene oxide (GO)-based drug delivery system (GDDS). However, current design of GDDS primarily depends on the prior experimental experience with the trial-and-error method. Thus, it is more appealing to theoretically predict possible GDDS candidates before experiments. Toward this end, we propose to fuse quantum genetic algorithm (QGA) and quantum mechanics (QM)/semi-empirical quantum mechanics (SQM)/force field (FF) to globally search the optimal binding interaction between the graphene/GO and drug in a given GDDS and develop a free computational platform “e-Graphene” to automatically predict/screen potential GDDS candidates. To make this platform more pragmatic for the rapid yet relatively accurate prediction, we further propose a cascade protocol via firstly conducting a fast QGA/FF calculation with fine QGA parameters and automatically passing the best chromosomes from QGA/FF to initialize a higher level QGA/SQM or QGA/QM calculation with coarse QGA parameters . By harnessing this platform and protocol, systematic tests on a typical GDDS containing an anticancer drug SN38 illustrate that high fabrication rates of hydroxyl, epoxy, and carboxyl groups on a pristine graphene model will compromise the stability of GDDS, implying that an appropriate functionalization rate is crucial for the delicate balance between the stability and solubility/biocompatibility of GDDS. Moreover, automatic GDDS screen in the DrugBank database is performed and elicits four potential GDDS candidates with enhanced stability than the commonly tested GDDS containing SN38 from the computational point of view. We hope that this work can provide a useful program and protocol for experimental scientists to rationally design/screen promising GDDS candidates prior to experimental tests.Graphene, as a novel category of carbon nanomaterials, has attracted a great attention in the field of drug delivery. Due to its large dual surface area, graphene can efficiently load drug molecules with high capacity  Pristine graphene, discovered by Geim and Novoselov, is a two-dimensional (2D) nanomaterial consisting of a single layer of carbon atoms  are surging and summarized in the comprehensive reviews  simulation and quantum mechanics (QM) programs are powerful to accurately investigate the non-bonded interaction between the graphene/GO and ligand for a given GDDS    generate graphene/GO models with different sizes and shapes, which can be intuitively visualized in three-dimensional (3D) viewer of e-Graphene; (2) inspect whether graphene/GO models satisfy the Hückel's rule (4n + 2) and full connectivity of Pi-bonds; (3) remove the duplicated GO models; (4) minimize graphene/GO models or ligands by the use of the natively implemented Tripos FF or external QM/SQM programs; (5) develop GDDS prediction/screen functions based on QGA and FF/SQM/QM; (6) implement an automatic and efficient cascade protocol for the GDDS prediction/screen; and (7) visualize GDDS prediction/screen results . For the convenience of users, four options for the automatic generation of a pristine graphene model have been implemented in e-Graphene.To improve the biocompatibility and solubility, pristine graphene is usually subjected to the chemical modification to become the GO material. However, the precise control of exact functionalization sites/rates on the pristine graphene is still quite challenging in the experiment. Therefore, an ensemble of GO models is generated to approximately stand for the fabricated GO material in the computational study. In our current implementation, common chemical modification groups  can be automatically added to a pristine graphene model. Furthermore, the number of decoration groups , functionalization direction , modification manner (random/systematic manner), and maximum number of GO models  can be sAn ensemble of GO models can be automatically enumerated in a random or systematic manner by e-Graphene; however, some produced GO models are actually the same structure after certain translations and rotations. Thus, it is necessary to remove those duplicated GO models from the ensemble before subsequent calculations. In this work, we firstly calculate a large-diameter circular fingerprint “2048bit-ECFP10,” which is one type of commonly used extended connectivity fingerprint (ECFP)  for a small molecule are automatically detected and recorded, and all the torsion angles of the freely rotatable bonds, besides the overall translation (three variables) and rotation (quaternions with four variables) of the small molecule, are firstly encoded in a chromosome with the form of real number. Then this real-coded chromosome, reflecting one specific conformation of the small molecule, is further converted to a binary-coded chromosome for the subsequent QGA.2+|β|2=1, where α and β refer to the probability amplitudes of a qubit (2 (or |β|2). If |α|2 (or |cosθ|2) is larger than |β|2 (or |sinθ|2), the qubit will be degraded to the ground state |0>, otherwise, it will be collapsed to the ground state |1>. Therefore, the concept “qubit” introduced in QGA provides the vast searching space.In QGA, each bit in each chromosome is referred to as a qubit, which is the basic unit of information in a quantum computer, and the corresponding state of each qubit is formulated by Dirac notation |ϕ > = α|0> +β|1> with the constraint of |α| a qubit . For thecosθ, sinθ) in each chromosome is initialized to , indicating that all the qubits initially have the same probability of being collapsed to either ground state |0> or |1>. If a randomly generated floating number between 0.0 and 1.0 is less than | cos(π/4) |2, the qubit will be degraded to the bit “1” (or |1>), otherwise, it will be collapsed to the bit “0” (or |0>). After the measurement of all the qubits in a chromosome, each chromosome is transformed to a conventional binary string containing only the bit “1/0,” which can be further decoded for the fitness calculation to update each chromosome for the next round of evolution.Before the evolution of each chromosome, each qubit (Quantum rotation gate operator (QRGO) is utilized to update each chromosome with the rotation angle (Δθ) by comparing with the best chromosome in the whole population  or binding energy between the graphene (or GO) and ligand by Equations . The potential energy or binding energy is computed by using the natively implemented Tripos FF in e-Graphene for the fast prediction or evoking the external QM/SQM programs for more accurate prediction.EGDDS, EG, and Elig are the potential energies of a given GDDS conformation, graphene/GO, and ligand, respectively. EDelta refers to the binding energy between the graphene/GO and ligand in a given GDDS conformation. The potential energy can be calculated by the internally implemented Tripos FF or externally evoked SQM/QM programs, such as Gaussian03/09/16, ORCA4.2, MOPAC2009/2012/2016, and XTB6.3. It is worth noting that Equation (5) is used to calculate the fitness by default, although both options  are supported in e-Graphene.where QGA run can be terminated if the maximum number of evolution generation is reached or the average fitness difference between certain successive steps are consistently lower than 0.001, which is the default convergence criterion and can be customized by users. Once completed, all the best chromosomes are decoded to return the final conformations with customized Tripos Mol2 format files containing the energy information, which can be visualized by another module in e-Graphene as shown below.To make e-Graphene more efficient, we further develop a cascade protocol for the GDDS prediction/screen. Even though the default two-layer cascade protocol (QGA/FF-QGA/SQM or QGA/FF-QGA/QM) is strongly recommended and extensively evaluated in this work, more computationally expensive three-layer (QGA/FF–QGA/SQM-QGA/QM) cascade protocol is also supported in e-Graphene program . Users cAfter GDDS prediction/screen, we are usually very interested in the optimal binding mode/energy between the graphene/GO and ligand for a given GDDS. For this purpose, we have implemented a convenient function to intuitively and synchronously visualize the graphene (or GO)-ligand binding mode/energy, and box information in 3D viewer of e-Graphene  so that https://www.dropbox.com/sh/xj8wnc08kmw8b7a/AAAyXFbObUsk28QwFVfLjXCka?dl=0) and (https://pan.baidu.com/s/15_N7FNPrfjjoaA8f2s5gwQ with the four-letter extraction code: urdf). It is worth mentioning that e-Graphene has been fully tested and directly run on the local machine with 64-bit version of Windows10 operating system (OS) and can also prepare GDDS prediction/screen jobs including the input files, scripts, and a binary package for a remote computer with 64-bit version of Redhat5/6/7, CentOS5/6/7, or Ubuntu16.04/18.04/20.04-LTS OS. To demonstrate the applicability of this program and protocol, two typical examples are provided as below.In this work, we propose to combine QGA and QM/SQM/FF for the intelligent prediction/screen of GDDS and further put forward an efficient cascade protocol by seamlessly integrating consecutive runs of QGA/FF–QGA/SQM (or QGA/FF–QGA/SQM-QGA/QM). For the convenience of users, we have developed a relatively comprehensive program “e-Graphene” with a graphic user interface to prepare and perform automatic prediction/screen of GDDS with/without the cascade protocol. The whole package including program, manual, and tutorial files is freely available on the Dropbox and Baidu Cloud public shared-folders with the respective download links  to produce five sets of 20 GO models, which represent five different rates of chemical modification on the pristine graphene model and are simply denoted by GO-1, GO-2, GO-3, GO-4, and GO-5, respectively, for the convenience of following discussion. Subsequently, one pristine graphene model and 100 GO models are combined to form a graphene/GO model database, which is systematically minimized in batch by evoking an external and efficient SQM program XTB6.3 with the keyword “–opt –gfn 2 –gbsa water reference” including the solvation and dispersion effect. Similarly, the 3D structure of SN38 with the neutral form is retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and also optimized by XTB6.3 with the same keyword.To tentatively explore this problem from the computational perspective, appropriate models should be produced to stand for the pristine and fabricated graphene materials. More concretely, a circular graphene model with the diameter of 21.760 Å and the total atom number of 168 is generated the menu , and thegfn 2 –gbsa water reference” including the solvation and dispersion effect as well. The respective probabilities of QRGO, QNGO, and crossover operation are set to 0.5, 0.1, and 0.7, respectively. Catastrophe operator is applied if the average fitness difference between two consecutive generations is always lower than 0.001 within 50 successive steps during the QGA evolution. The fitness is calculated by Equation (5) for the current test. According to these parameters, this cascade protocol was repeated three times for each GDDS because of the intrinsic randomness in QGA. Therefore, 303 runs with this cascade protocol were conducted, and all the corresponding results for the best chromosomes are listed in In order to investigate the effect of different graphene-functionalization rates and sites on the stability of GDDS including SN38, the two-layer cascade protocol (QGA/FF–QGA/SQM) will be applied to all 101 graphene/GO-SN38 pairs due to our limited computational resource. In this protocol, the full parameter settings for current tests are shown in Before evaluating the impact of different functionalization rates and sites on the stability of GDDS, the convergence analysis was first conducted to inspect whether QGA/FF in our cascade protocol could assist the convergence of QGA/SQM. For the convenience of discussion, one QGA run for the GDDS containing the pristine graphene model and SN38 was taken as an example. The average fitness of the whole chromosomes along the evolution of QGA/SQM is monitored because our final optimal solutions are actually derived from QGA/SQM rather than QGA/FF. In To scrutinize the influence of different functionalization rates on the GDDS stability, the binding energy between the graphene/GO model and SN38 is derived from the best chromosome after running the two-layer (QGA/FF–QGA/SQM) cascade protocol. All the results are listed in To further evaluate the impact of different functionalization sites on the stability of GDDS with the same fabrication rate, the histogram of binding energies between the GO models and SN38 is as plotted in opt –gfn 2 –gbsa water reference,” which are finally utilized for the current screen by the two-layer cascade protocol (QGA/FF–QGA/SQM). SQM is also set to XTB6.3 with the keyword “–gfn 2 –gbsa water reference” that takes into account the solvation and dispersion effect. All the parameters for this protocol used in the screen are exactly the same as those in the previous section. This screen campaign was also performed for three times due to the inherent stochastic nature of QGA.Our cascade protocol can also be used to computationally screen the potential GDDS candidates for a given graphene (or GO) model and a given chemical database. For the demonstration purpose, we adopt the same circular pristine graphene model (diameter: 21.760 Å) as mentioned above and choose the DrugBank database  QGA belongs to the stochastic method, which produces different results for different runs. Thus, multiple runs are strongly recommended to achieve more convergent results, which undoubtedly brings some extra computational burden. (2) The speed and accuracy of two-layer (QGA/FF–QGA/SQM) or three-layer (QGA/FF–QGA/SQM–QGA/QM) cascade protocol was largely dependent on the computationally expensive SQM or QM with different keywords and parameters. Careful benchmarks of different choices are highly advised to obtain the comparatively accurate yet fast combination for the practical prediction/screen of GDDS. Thus, it is suggested for users to clearly understand the weakness of e-Graphene before applying it to their own projects.As recent booming of quantum computation in various areas, QGA possesses intrinsic quantum operators, thus we tentatively envision that if QGA can be ported to the quantum computer simulator and subsequent real quantum computer, QGA might release its tremendous native capacity. However, it still has a very long way to go before QGA-based protocol can be fully deployed in the quantum computer due to the following two main obstacles: (1) the current quantum computers support a small number of qubits, e.g., 53 bits from Google quantum computer (Arute et al., In this work, we present a practical computational platform “e-Graphene” for the automatic prediction/screen of GDDS by the seamless integration between QGA and QM/SQM/FF and further propose a cascade protocol for the efficient prediction/screen of GDDS. In this platform, a graphene/GO model can be automatically generated according to the shape and size specified by users, subsequently minimized by the internally implemented Tripos FF or externally evoked QM/SQM programs, and finally used for the preparation of GDDS prediction/screen job with/without the cascade protocol, which can be ran on the local machine with Windows10 and on the remote machine with Red Hat/CentOS/Ubuntu. By using this platform, two representative test cases are adopted to demonstrate its pragmatic functions. One is systematic tests on a extensively used GDDS containing an anticancer drug SN38, which manifests that high rates of functionalization on the pristine graphene reduces the stability of GDDS and also indicates that an appropriate fabrication rate is crucial to attain an optimal GDDS with good stability, solubility, and biocompatibility. The other is the GDDS screen in the DrugBank database for a pristine graphene model, which harvests four potential GDDS candidates with better stability than the common GDDS containing SN38. At last, the limitations of this computational platform are also addressed, although it is envisioned that this free platform could enable experimental scientists to automatically predict possible GDDS candidates before experimental tests.The original contributions presented in the study are included in the article/SZ and JX performed the calculations and analysis. LW and DZ tested the program. YX and FL revised the manuscript. SZ and FL conceived the workflow, wrote the manuscript, and developed the program. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The synthesis of Graphene is critical to achieving its functions in practical applications. Different methods have been used to synthesis graphene, but graphite exfoliation is considered the simplest way to produce graphene and graphene oxide. In general, controlling the synthesis conditions to achieving the optimum yield, keeping the pristine structure to realize on-demand properties, minimum layers with the smallest lateral size, and minimum oxygen content are the most obstacles experienced by researchers. Each application requires a specific graphene model, graphene oxides GO, or even graphene intercalated compounds (GIC) depending on synthesis conditions and approach. This paper reviewed and summarized the most researches in this field and focusing on exfoliation methods. It is known that graphite and diamond are the two natural crystalline allotropic forms of carbon for a prolonged time. The atomic arrangement of carbon atoms in these materials gives different properties. For example, graphite is soft and black, and stable, while diamond is hard and transparent [1]. Diamond and graphite consist of extended networks of spAllotropes are elements that are chemically identical but vary in their physical properties. In 1985, the discovery of fullerenes by Kroto et al. [3] marked the beginning of an era of synthetic carbon allotropes with striking properties. Iijima, in 1991, discovered carbon nanotube (CNT) [4]. While in 2004, graphene, which is a two-dimensional form of graphite, was isolated at Manchester University by Novoselov et al. [5]. 2 graphitic materials, including (0D) fullerenes, (1D) carbon nanotubes, and stacked into (3D) graphite [7].Graphene is a 2-dimensional structure consists of a single atom thick sheet of carbon crystallized as a honeycomb structure monolayer [6]. Graphene has been considered as the fundamental building block for all spn . The physical, chemical and mechanical properties of the allotropes of carbon  are different depending on valence bond hybridization [8].Allotropes of carbon can be defined by valence bonds hybridizations spGraphite has a layered planar structure wherein each carbon atoms layer is arranged in a honeycomb lattice with a separation of 0.142 nm. The interplanar spacing, d, is (0.335nm) [9]. In diamond, the carbon atoms are arranged in a face-centered cubic crystal structure (FCC) system with diamond lattice [10]. Fullerenes are zero-dimensional hollow carbon cage structures with carbon atoms arranged in a lattice similar to the soccer-ball. The most widely publicized fullerene is buckyball (C60). Carbon nanotubes (CNTs) are one dimension structure that can be viewed as a hollow cylinder formed by rolling a two-dimensional graphene sheet into a cylinder with a half fullerene at its ends. Carbon nanotubes are either single-walled CNTs (SWNT) or multiwalled CNTs (MWNT) [11]. Graphene is a flat single atom thick sheet of carbon packed into a honeycomb crystal plane and has a 2D structure [6]. Due to the outstanding features and large applications, Graphene has attracted great interest from scientists and engineers [7]. Moreover, it is the thinnest and strongest material that is ever subjected to measurement [12]. Graphite is considered to consist of huge numbers of graphene layers. There are two kinds of bonds form among the graphene layers. The graphene layers are held together by the weak Vander Waal attraction with a length of about 0.341nm between the adjacent graphene layers [13]. This weak attraction between layers can ease the exfoliation into individual layers. The other bond is between carbon-carbon atoms with 0.142 nm spacing, which is a strong covalent bond entire each layer [14]. Graphenes can exist in different forms: graphene oxide, chemically modified graphenes, bilayer graphenes, etc. These nanomaterials have many medical applications, integrated circuits, transparent conducting electrodes, desalination, solar cells, energy storage, biodevices, etc. [15].2/V.S) [18], which means an electron moves through it without much scattering or resistance. Graphene has a high specific surface area of 2630 m2/g [19]. The unique structure enables graphene of many distinctive properties and promising applications. Graphene is the strongest material ever measured and has excellent mechanical properties with an elastic modulus of 1 TPa and ultimate tensile strength of 130 GPa [20]. Thus, graphene is the strongest material ever discovered to date. It is estimated that graphene is 100 times stronger than the best steel ever for the same thickness [17]. Thus, graphene is a promising material for high-performance nanoelectronics, heat dissipation, sensors, field emission, and transparent conductor. One single-layer of graphene has a transparency of 97.7% of incident light and absorbs only 2.3% and, it is almost completely transparent [21]. Due to its unique thermal, electrical, and mechanical characteristics, Graphene has been utilized in many applications. A Single-layer of graphene has thermal conductivity as high as 5000 W/m.K [16] and higher about 10 times than Cu and superior to carbon nanotubes (CNTs) and diamond [17]. Graphene has high mobility of charge carriers , and electrochemical are the most commonly used methods.4.1. Chemical vapour deposition (CVD)The chemical vapor deposition (CVD) on transition metals  is another method used to prepare graphene, which requires a temperature as high as 1000 °C with a hydrocarbon gas flow as precursor . This method is applied at high temperatures when exposing transition metal to low concentration hydrocarbon gas till the surface is saturated with carbon atoms  .Methane gas is generally used as a source of carbon. Hydrogen and argon are also used with methane as reaction stabilizers and enhancing the film uniformity. Zhan et al. in 2018 [40] showed a strong correlation between substrate morphology and graphene domain density. The use of a pre-annealed Cu foil followed by electro-polishing treatment will give ultra-smooth Cu surfaces. The graphene grows on annealed electro-polished Cu substrates show better quality in terms of lower domain density and higher layer uniformity than those grown on Cu substrates with only annealing or only electro-polishing treatment. Well-ordered graphite films of nanometer thickness have been grown on Ni substrates using chemical vapor deposition (CVD) with a thickness of a few graphene layers with graphite film thickness is about 1.5 ± 0.5 nm [41].Ni as the catalyst has some limitations such that an over a small region of few to tens of microns, single and few-layered graphene is obtained and not homogeneously throughout the entire substrate. The control lack over the number of the layers is partial because, upon cooling, the carbon segregation from the metal carbide happens rapidly within the nickel grains and heterogeneously at the grain boundaries. The use of polycrystalline copper foils as a substrate in CVD gave exceptional results in uniform deposition of high-quality single-layered graphene over large areas [30]. This method can produce high-quality graphene and suitable for mass production, but it is not cost-effective and easy. The availability of high-quality and large-area graphene is a crucial requirement for the production of graphene-based nanoelectronics. As discussed above, the other methods for graphene production have their limitation. The CVD method [38] requires a purification process to eliminate the catalyst particles and transfer Graphene to another substrate; the chemical reduction of GO requires chemicals, which can affect graphene properties; the scotch tape method [5] is not suitable for large scale production of Graphene. The field of epitaxial graphene on silicon carbide is rapidly growing by annealing SiC as an insulator at high temperatures under vacuum to form a graphitic surface by sublimating Si atoms [42]  [43]. The potential of epitaxial graphene as new electronic material is now well recognized [44]. Silicon carbide (SiC) was used as substrates for the epitaxial growth of graphene. The substrate is heated at high temperatures (>1250 °C) and low pressures (~10−6 Torr). Ultrathin epitaxial graphite films were grown by thermal decomposition on the (0001) surface of 6H-SiC. The films are typically composed of 3 graphene sheets [45]. The graphene epitaxial growth on silicon carbide is considered an ideal material for high-end electronics that might surpass silicon in terms of key parameters such as speed, feature size, and power consumption [44]. The epitaxial growth method can prepare high-quality graphene with excellent properties, but it is not easy to obtain a high production rate with this method.Kretschmer et al. [46] were the first that utilized the arc-discharge method to synthesize C60 [47]. The main concept of graphene synthesis by arc discharge is similar when applying an electric field between two graphite electrodes under argon or helium gases. When the electrodes’ distance between the two electrodes becomes close to 1–2 mm, the arc discharge starts, and the evaporated materials from the anode are deposited on the cathode. The anode is almost previously compounded with catalysts . Also, the cathode should be rotated to keep the distance constant [48]. The arc discharge process is illustrated in Figure 4.2SO4 (90%) /HNO3 (70%) [49]. In comparison, Kosynkin et al. (2009) obtained oxidized graphene nanoribbons by suspending MWCNTs in concentrated H2SO4 and then treated with KMnO4. The isolated graphene nanoribbons were highly soluble in water, ethanol, and other polar organic solvents [50]. The nanotubes’ unzipping seems similar to the ‘unzipping’ of graphite oxide [51] and [52]. The opening could occur longitudinally or spirally way, depending on the attacked site and chirality angle. Manganate ester formation (O=MnO-2=O) is the main reason to destroy the alkene structure of CNT, making these initial attacked sites more exposable to the next intensive reaction with permanganate [50]. Figure 5 illustrates the proposed unzipping mechanism of CNTs [50]. Saito et al. (2002) showed that MWCNTs could be cut to several nanometers of CNTs by sonication in mixed strong acids (H4.5.1. Graphene oxide3 and fuming nitric HNO3 + NO2. Staudenmaier developed this method later in 1898 [54]. Another improvement in the Brodie method was made by Hofmann and König (1937) [55]. Hummers and Offeman made further development on the Brodie method [56]. Marcano et al. [57] improved on the Hummers method by eliminating sodium nitrate, thus excluding toxic nitrous gas production.Large amounts of graphene can be produced at a reasonable cost from graphite oxide. Graphene oxide can be prepared by oxidation of graphite using concentrated acids and strong oxidants . The oxidation of graphite to form graphite oxide was introduced in 1859 by Brodie [53] when graphite was oxidized in the presence of potassium chlorate KClOIn these modifications, the key developments concentrate on the use of safer oxidants and more effective procedures. Staudenmaier and Hofmann’s methods use potassium chlorate, and the methods of Hummers and Tour use potassium permanganate as the primary oxidant. Nitric, sulphuric and phosphoric acids or their mixtures are used as solvents in both synthesis methods.4, NaNO3, and H2SO4 to oxidize graphite into graphite oxide within a few hours. The advantages of Hummer’s method are as follows. First, the reaction is completed within a few hours. Second, the oxidizing agent KMnO4 is used to replace KClO3 and improving reaction safety by avoiding the evolution of explosive ClO2 and KClO3. Third, the use of NaNO3 to replace HNO3 eliminates the forming of acid fog [58].Brodie was the first to prepare graphite oxide by oxidation of graphite in a mixture of potassium chlorate. An oxidizing agent and nitric acid helped to intercalate graphite and expand it [53]. Later on, Staudenmaier et al. [54] used nitric acid: sulphuric acid with a volume ratio of 1:4 in an ice bath. Then, graphite was added (4.37 g for every 100 mL) with continuous stirring. The oxidizing agent is potassium chlorate. The weight ratio of graphite to the oxidizing agent is 1:11. Staudenmaier’s method is considered safer than the Brodie method, but toxic gasses are still created; this is why using the Hummers method more than Staudenmaier and Brodie method. In 1958, Hummer developed a new method [56], which is the most common method for preparing graphene oxide using KMnO2, N2O4). In this method, sodium nitrate (NaNO3) was replaced by potassium permanganate. The mixture of reactions consists of (9:1) H2SO4: H3PO4 for the oxidation reaction [57].Later on, a new method was developed by Tour and his group in 2010 to improve the Hummers method in the degree of GO oxidation as well as to minimize the generation of toxic gases (NO2- and sp3- hybridized carbon (C) atoms decorated with oxygen-containing functionalities. These functional groups include hydroxyl (C–OH) and epoxide (C–O–C) functional groups on the basal plane and carbonyl (C=O)) and carboxyl (COOH) groups at the sheet edges [60]. These functionality groups introduce sp3 defect sites to the nanosheets, distorting the intrinsic conjugated π system and lowering overall strength and conductivity. The saturated sp3 carbon atoms bound to oxygen make GO an insulator. Graphene oxide is an electronically hybrid substance that displays both a large energy gap between the σ-states of its sp3- bonded carbons and conducting π-states from sp2 carbon sites [61]. By reducing chemistry, the tunability of the ratio of the sp2 and sp3 fractions is a powerful way to tune its bandgap and, thus, controllably transform graphene oxide from an insulator to a semiconductor or semi-metal [62].Graphene oxide is a single layer of graphite oxide. The chemical oxidation of graphite and its subsequent exfoliation in water results in single-layered GO sheets [59]. Graphene oxide contains a mixture of spWith the recovery of a conjugated structure, graphene oxide can be reduced to graphene by eliminating the oxygen-containing groups. Reduced GO (rGO) sheets are generally referred to as chemically modified graphene, chemically transformed graphene, or reduced graphene oxide [62].2 conjugated graphene network [62]. In thermal or chemical reduction, the functional groups C=O and O=C-OH could be partially removed or converted to a new chemical species such as (C-OH). The degradation of the electrical properties of the RGO is due to the remaining functional groups C-OH or C-O-C in RGO that could not be reduced easily [66].Graphene oxide is an electrically insulating material but can be converted to a conductor by chemical reduction process, electrochemical reduction, thermal treatment, and green reduction , as shown in Figure 7. However, none of the reported reduction methods yield complete oxygen removal, resulting in only partial restoration of the sp2 gases. These gases create large pressure between graphene oxide stacked layers, which are enough to exfoliate individual GO layers is enough to separate two stacked GO sheets [67].Thermal reduction of GO was made by conventional heating  [67], microwave [68], and photo-reduction [69]. In thermal annealing, GO is heated to a high temperature under inert gases such as argon gas. The rapid increase in temperature makes oxygen functional groups decompose into CO and COMicrowave irradiation (MWI) is another thermal reduction method of GO using a microwave oven. This is a rapid method and gives uniform heating of the GO [68]. Zhang et al. proposed another method: a photo-reduction of GO using femtosecond laser irradiation [69].2NH2.xH2O) and dimethyl-hydrazine, can be used as a chemical reducing agent for GO.Another method of GO reduction is the chemical method, which can be carried out using a chemical reducing agent. This is an effective method for mass production of graphene because of availability and low cost compared with other reduction methods [65]. Stankovich et al. were the first to prepare reduced graphene oxide (rGO) [36] using hydrazine as a chemical reducing agent. Hydrazine and its derivatives, such as hydrazine hydrate  by donation of electrons with acceptance of oxygen while it was converted into an intermediate oxidized polymerized thiophene that losses sulfur atoms and transformed into polyhydrocarbon that heat-treated to obtained reduced GO [71]. M. J. Fernandez-Merino et al. used different reducing agents sodium borohydride, pyrogallol, and vitamin C, in addition to hydrazine and made a comparison among them. They found that vitamin C has highest yield suspension compared with other compounds [72]. T.T. Dang et al. reported the influence of temperature on the reduction of graphene oxide by hydrazine in N, N-dimethylformamide (DMF)/HR. Tarcan et al. reduced graphene oxide in N, N-dimethylformamide without using any reducing agents using the solvothermal microwave-assisted method. So, this method gives stable and highly concentrated dispersions of GO through partial removal of oxygenated groups from graphene oxide surface [74].The electrochemical reduction of GO relies on removing oxygen functional groups using regular electrochemical cells containing aqueous solutions (electrolyte) at ambient temperature . Graphene oxide reduction occurs by the exchange of electrons between the inert electrodes and GO sheets or films. This method eliminates the usage of dangerous reducing agents as hydrazine.The green synthesis approach of graphene reduction is rapidly improved due to its low cost, ease of handling. It is an eco-friendly method without hazards, harsh, and expensive materials [77]. Biomolecules, microbes, and plant extracts are used to reduce and cap agents widely used to synthesize metal nanoparticles. Generally, plants contain a wide variety of bio-molecular substances, including amino acids, proteins, alcoholics, polysaccharides, enzymes, polyphenols, and vitamins. Plant extracts may also be used for graph synthesis as a reduction and stabilization agent . Ascorbic acid or (Vitamin C) is a recently reported reducing reagent for graphene oxide with benefits over hydrazine [72]. Ascorbic acid is nontoxic and has higher chemical stability with water so that the produced RGO does not result in aggregation. Taking into consideration the environment and human health in recent years, reduced graphene oxides (RGOs) were produced by green reduction of graphene oxide (GO) using ascorbic acid [72], glucose [80], fructose and sucrose [68], tea leaves [77], melatonin [79] and alcohols [81].2 in size [83]. This method is called the Scotch tape approach, which is not suitable for large production of graphene.Geim and Novoselov [7] prepared a single graphene sheet by peeling off a sheet of graphite using Scotch tape  [82]. This method involves repeatedly peeling highly oriented pyrolytic graphite (HOPG) using scotch tape. The process has been optimized to produce single-layer graphene (SLG) with high structural quality and more than 100 μm2/g), and enhanced surface activity [84]. The increase in the surface area of material can enhance their chemical and physical reactivity. This is very vital in many applications where the surface activity is important such as catalytic materials, ion exchange [85], fillers materials in composites [86], intumescent  material [84].Layered materials such as graphite consist of two-dimensional platelets weakly stacked to form three-dimensional structures. In graphite, these layers form strong chemical bonds in-plane but display weak out-of-plane bonding. This facilitates the exfoliation process to form so-called nanosheets (about micrometers wide but less than a nanometer thick). Such exfoliation leads to an increase in the material’s surface area, over 1000  is a top-down method of exfoliation of natural graphite into few-layer graphenes dispersed in liquid media [87] and are becoming more desirable because they are cost-efficient, using inexpensive graphite, scalable process, produce defect-free graphene, and can be used to deposit the dispersed graphene in a variety of environments and on different substrates not available in using mechanical cleavage or growth methods [88–90].This process involves graphite dispersal in a solvent, ultrasound exfoliation or other mechanical dispersal procedures, and purification. The involved mechanisms of fragmentation and exfoliation have usually been attributed to the force induced by ultrasound and the interaction with the solvent molecules . Liquid phase exfoliation is a four-step method as illustrated by Shen and collaborators [93–95] that include: 1) graphite immersion into the liquid medium, 2) intercalation of the graphite, 3) the exfoliation of these planes, and 4) the stabilization of the exfoliated materials isolated in the medium. In step one, the liquid medium for graphite immersion should effectively reduce van der Waals forces’ attraction between the layers of the nonexfoliated material. The liquid molecules must be inserted between the layers of the crystal before exfoliation. During the intercalation step of the graphite, the interaction between the graphite graphene planes is decreased. In the third step, the exfoliation of these planes occurs, which can be mechanically assisted or not. When 2D material with the liquid molecules inserted is exposed to a mechanical fragmentation force such as shear, van der Waals’ attractive forces can be overcome, facilitating the individual layers’ exfoliation. Finally, in the fourth step, the exfoliated material isolated in the liquid medium should be remaining stable. A fundamental characteristic of this method is the possibility of producing mono and few-layer graphenes stabilized in different media [93–95]. Different precursors of graphitic material are used in Liquid-phase exfoliation, such as graphite oxide, natural graphite, graphite intercalation compound (GIC), and sonication-free liquid-phase exfoliation. These graphite precursors are directly subjected to a solvent treatment to weaken the van der Waals attractive forces between graphene interlayers using external driving forces such as ultrasonication, electric field, shearing, or heat  [96].Liquid-phase exfoliation of graphite oxide is the most widely used method for the preparation of graphene. In this method, the graphite is intercalated with strong oxidizing agents , followed by graphite layers’ expansion via sonication. The reduction of the obtained graphene oxide to graphene is usually conducted by either thermal or chemical approaches [36]. To exfoliate, it is important to resolve the attractions of the van der Waals between adjacent layers. One way to reduce these attractions by oxidation and chemical intercalating reactions is to increase the distance between adjacent layers .The oxygen functional groups such as hydroxyl and epoxy are inserted and attached to graphite layers during graphite oxidation. The d-spacing between these layers has increased significantly from 3.4 to around 7.0 A .The hydroxyl or carboxyl groups covalently bonded with graphene oxide make graphene oxide dispersable in some organic solvents at concentrations of up to 1 mg/mL [36] and water up to 7 mg/ mL [99]. However, the oxidization process significantly causes defects in graphene. Stankovich et al. [36] used hydrazine to reduce GO, and the chemically reduced graphene product (CRG) still has some defects. Other reduction methods are as follows: the solvothermal reduction method of GO in N, N-dimethylformamide (DMF) with hydrazine [100] or without hydrazine [101] as a reducing agent, the use of metal Fe to reduce exfoliated GO [102]. None of these reduction methods can recover the graphene structure completely, and some oxygen-containing groups are still irremovable.The liquid phase exfoliation method and exfoliation of GO of graphite are technically similar, but the first does not involve the oxidation step. Graphene flakes can be produced by exfoliation of graphite using chemical wet dispersion followed by ultrasonication in water and organic solvents. This method’s principal attraction is a simple and scalable process where pristine graphite or expandable graphite is subjected to a solvent treatment to weaken the van der Waals attractive forces between graphene interlayers. External driving forces such as ultrasonication, shearing, or electric field can be applied to help exfoliation into graphene [92]. Graphene’s exfoliation occurs because of the strong interactions between the solvent molecules and the graphitic basal planes, overcoming the energetic penalty for exfoliation and subsequent dispersion. For successful exfoliation, overcoming the van der Waals attractions between the adjacent layers of graphite is necessary.Graphite intercalation compounds (GICs) are formed by the insertion of atomic or molecular layers of a different chemical species  called the intercalant between host graphite sheets [103], see Figure 10. The graphite intercalation compound (GICs), also known as the expanded graphite, is a synthesized intercalation compound of graphite that expands or exfoliates when heated. The intercalation increases the distance between the graphite layers. In pristine graphite, the distance between the layers is typically 0.34 nm, while in a potassium-GIC, the sheet distance has been measured to about 0.53 nm [103]. Because of the increased distance between the graphite layers in the GIC, the van der Waals attraction between the layers was weakened. Thus, the layers can be more easily separated to acquire the desired graphene [104]. The number of layers separated by an intercalant layer is defined as staging. The stage index n denotes the number of graphite layers between adjacent intercalate layers of structural ordering. Fully intercalated graphite will be a stage 1 GIC [105]. The graphite intercalation compounds are of particular physical interest because they have a relatively high structural ordering degree. Although graphene is a material with a single carbon layer, the definition has been broadened in recent years, and the term ‘’few-layer graphene’’ is often used. If the number of carbon layers is 10 or more, the piece is known as a graphite nanoplatelet (GNP), also known as an exfoliated graphite nanoplatelet (GNP) or a graphite nanosheet (GNS) [106].Güler et al. 2016 [107] prepared multilayer graphene by mixing hexagonal graphite (HG) with an acidic mixture to form graphite- intercalated compound (GIC). The GIC is then heated at a temperature between 800–900 ºC to form expanded graphite (EG), followed by dispersion in an organic solvent through ultrasonication.The exfoliated graphite exhibited excellent properties of flexibility, lubricity, and adsorption. The exfoliated graphite has many applications in various fields, such as absorption of spilled oil in water , sealing material [110], fire retardant [111], and electrode [112]. 2 . Graphite’s surface energy is the minimum energy per unit area sufficient for the van der Waals force when two sheets are peeling apart. Graphene and solvent surface energies have to be very close for a successful exfoliation process. The enthalpy of mixing will be reduced, and the exfoliation will increase. Liquids with surface tensions of about 30–40 mJ/m2 are typically good solvent candidates for graphene exfoliation, such as N-methyl-2-pyrrolidone (NMP 40 mJ/m2) [33], N, N-dimethylformamide (DMF 37.1 mJ/m2) [113].Graphite can be exfoliated into single-layer graphene (SLG) and few-layer graphene (FLG) in a solvent having a surface tension of about 40 mJ/mSonication-assisted liquid-phase exfoliation of graphite to give Graphene was first reported by Hernandez in 2008 [33]. In their work, graphite powder was dispersed in a specified organic solvent, such as N, N-dimethylformamide (DMF), and N-methyl pyrrolidone (NMP), followed by sonication and centrifugation. The LPE yield is estimated by measuring graphene concentration in the dispersion using UV-Vis spectroscopy and exploiting the Beer–Lambert law. The degree of exfoliation was determined by analyzing the number of layers in a graphene flake using TEM, AFM, and/or Raman spectroscopy. Defect-free monolayer graphene, bi-layer, and multilayer graphene are obtained. The solubility of graphene in the dispersion was 0.01 mg/mL after 30 min sonication, centrifugation at 500 rpm for 90 min. The number fraction of monolayer graphene in NMP dispersions was estimated at 28%. It was found that NMP gives the best thermodynamic stabilization due to the good matching of graphite –solvent surface energies. The most important criterion for successful graphite exfoliation is the good matching of solvent surface energies with graphene. The best solvent is that with surface energies close to the graphene surface energy [114–116].Graphite consists of many graphene layers joined together by van der Waals attractions between the adjacent layers. Successful exfoliation of graphite requires the overcoming of the van der Waals attractions forces between the adjacent layers. When graphite is immersed in a liquid, the interfacial tension between graphite and solvents should be equal to the graphene-graphene interaction energy [117]. A very close graphite- solvent surface energies resulted in small mixing enthalpy, and the exfoliation occurs more easily [118]. Good solvents are used to disperse graphene minimizing the interfacial tension between the solvent and graphene flakes. The liquid’s surface tension is the force required to resist an external force due to liquid molecules’ cohesive nature. If the interfacial tension between graphitic flakes and the solvent is high, there is poor dispersibility of the solvent’s graphitic flakes [114]. The flakes tend to adhere to each other due to the flakes’ cohesion force and hindering their dispersion in the solvent [119]. Hernandez et al.  reported that solvent with surface tension 40 mN/m is the best solvents for dispersing graphene and graphitic flakes. This will minimize the interfacial tension between the solvent and graphene [119]. It has to be noted that direct LPE in organic solvents like NMP normally produces graphene flakes with lateral dimensions typically < 1 μm, which are too small for applications such as mechanical reinforcement in composites.The solvent molecule-graphene interactions were investigated by Liu et al. [120] using electrophoresis and zeta potential measurements. The charge transfer between the graphene and solvent molecules is responsible for the graphene’s surface charge. Graphene can be positively or negatively charged in the dispersion exhibiting different zeta potential values depending on the different donor and acceptor numbers of a solvent. The electrostatic repulsion of charged graphene sheets will ease the stable dispersions. 2 are the solvents’ toxicity. NMP organic liquid is an eye irritant and also toxic to the reproductive organs [121]. DMF may have toxic effects on multiple organs [122].The main disadvantages of using a solvent with a surface tension of 30–40 mJ/mThe other disadvantages of this method are the low stability, low single-layer content (0.01 mg/mL), and the solvents’ high boiling points make it very difficult to remove the solvent after exfoliation [33]. Therefore, the search for additional solvents is highly recommended to overcome the low yield problem associated with graphene dispersions in organic solvents.Utilizing ionic liquids (ILs) in graphite exfoliation is considered green chemistry due to it is nontoxic, stable chemically, no need for vapor pressure, recyclable by distillation or ion change. Exfoliation by ionic liquids is also considered a one-step procedure (direct exfoliation). Ionic liquids give graphene unique properties, unlike chemical modification that destroys the graphene electronic structure. Ion liquids are completely organic or partially inorganic salts with a melting point below (100 °C). The most significant factor in this technique is the surface tension of ILs, which has to be very close to that of graphene .Fukushima et al. were the first to use ILs to untangle carbon nanotubes using imidazolium-based ILs as solvents assuming interactions between the positive charge of the imidazolium rings and p electrons of the nanotube [125]. Liu et al. prepared graphene nanosheets using imidazolium ILs assisted electrochemical synthesis [126]. Zhou et al. incorporated polymerized ionic liquid with 1-butyl-3-methylimidazolium hexafluorophosphate to disperse graphene. Nonoxidized few-layer graphene highly concentrated and stable colloidal (0.95 mg/mL) was prepared by ultrasonication of natural graphite flakes in this ionic liquid [127]. Nuvoli et al. prepared high concentration few-layer graphene sheets using LPE by grounding and then sonication of graphite in 1-hexyl-3-methylimidazolium hexafluorophosphate (HMIH) as an ionic liquid. The graphene concentration (up to 5.33 mg/ mL) is obtained. Ionic liquids can be useful when green chemistry and/or a nonvolatile, stable solvent medium is required [124].In 2014, Shang et al. prepared large quantities of solvent-free graphene nanosheets and nanodots with low oxygen content have by mechanical grinding exfoliation of natural graphite in a small quantity of ionic liquids  [128]. Electrochemical exfoliation of graphite rods was used to synthesis carbon dots (CDs) by dissolved cheap salts (NaCl and KCl) in distilled water as an electrolyte [129]. Generally, exfoliation using ionic liquids has been associated with other techniques as electrochemical exfoliation, mechanical grinding, or sonication to be more producible.Liquid phase exfoliation by a surfactant is considered an ideal method to prepare high stable concentrated graphene dispersion despite the surfactant’s defects [130]. Six surfactants were utilized involving ionic and nonionic: sodium deoxycholate SDBS, sodium dodecyl sulfate HTAB, Ssodium dodecyl benzenesulfonate SDS, hexadecyltrimethylammonium bromide, and Triton X-100 and Tween 80. The theory explains the different ionic and nonionic surfactants’ different mechanisms in stabilizing graphene dispersions for colloidal stability [130]. Commercially, there are around 20–30 available surfactants that have been used to exfoliate graphite. The main types of surfactants are cationic, anionic, and nonionic surfactants. Sodium dodecyl sulfate (SDS) is an anionic surfactant with a small molecule with a hydrophobic tail and a polar head group [131]. In addition to previous classes, pluronic and tetronic block copolymers were used to produce graphene dispersions with concentrations exceeding 0.07 mg mL-1 via sonication and centrifugation [132].Ionic surfactants such as cetyltrimethylammonium bromide (CTAB) [133], sodium dodecylbenzene sulfonate (SDBS) [33], sodium cholate (SDOC), and other surfactants [134] were used to exfoliate graphite flakes. Ultrasound treatment of graphite dispersion in N-methyl pyrrolidone solution yields a dispersion with a concentration of 2.21 mg/mL [135]. Graphene dispersed in a monomer (tetraethylene glycol diacrylate), and the latter’s subsequent polymerization yields a dispersion of 9.45 mg/mL [136]. For graphene dispersions in two organosilanes, 3-glycidoxypropyl trimethoxysilane (GPTMS) and phenyl triethoxysilane (PhTES), the concentrations were 0.66 and 8.00 mg/mL for PhTES and GPTMS, respectively [137]. Guardia et al. examined the differences between ionic and nonionic surfactants and showed nonionic surfactants’ dominance [138]. Niu et al. reported a low-cost and environmentally friendly method to directly exfoliate graphite powders into few-layer graphene sheets using inorganic salts to expand graphite for easy exfoliation by low-power ultrasonication [139]. The addition of ethanol to water/surfactant solutions can lead to a high concentration (∼0.46 mg/ml) of graphene dispersions. About 10 wt% of ethanol addition for ionic and nonionic surfactants was found to enhance the exfoliation efficiency and the graphene concentration three times. This enhancement is due to decreasing mixing enthalpy and enhancing the stability of surfactants .A simple and inexpensive green route for large-scale production of exfoliated graphene dispersions using direct sonication of graphene and aqueous solution of perylene tetracarboxylate (PTCA) aromatic semiconducting surfactant. This produced a high yield of single and few-layer graphene sheets with minimal basal plane defects [142]. A surfactant-assisted ultrasonic exfoliation of mildly oxidized graphite has been successfully demonstrated using cationic surfactants and one anionic surfactant. The surfactant charge was found to be a significant factor in affecting the exfoliation efficiency [143].One of graphene’s solvent exfoliation problems is that the good solvents tend to have high boiling points that present problems for flake deposition, so they also are difficult to remove. Low boiling point solvents such as chloroform, isopropanol, and 1-propanol were explored by many researchers for LPE production of graphene because of the removal difficulty of high boiling solvents like DMF NMP, etc. . Catheline et al. prepared individualized graphene flakes as single and double layers with one micron as average lateral size by dissolving the graphite intercalation compounds KC8 in low boiling points ether solvents such as methyl-tetrahydrofuran , tetrahydrofuran , and cyclopentyl methyl ether . This method is expected to significantly benefit composites and thinner film preparation due to high dispersity, stability, or double graphene levels [146].2SO4 solution. The exfoliated graphene sheets exhibit lateral size up to 30 μm [150]. Although this method is easy to implement and prepare graphene, the obtained graphene still has many oxygen functional groups and structural defects due to the oxidation reactions at the graphite anode. When a voltage is applied in a conductive solution for the graphite electrodes, the graphite electrodes can corrode and produce the solvent-functioning graphite nanosheets  [147]. The use of electrochemical exfoliation of graphite anode to prepare graphene was first reported by [126] using a mixed solution containing ionic liquid and water as the electrolyte. Structure with up to 10 graphene layers is called few-layer graphene, while the structure with more than 10, and less than 100 graphene layers is called thin-film graphite [148]. Liu and his group [149] believed that electrochemical exfoliation would play more critical roles in synthesizing graphene materials, but many exciting challenges are still waiting to be explored. These challenges include a better understanding of graphite exfoliation reaction mechanism, parameters that affect large-scale graphene production, such as complex fluid mixing, heat, and mass transport. Finally, the obtained graphene material is suitable for many applications with unique features. For example, in membrane applications, graphene materials must behave tailored size, interlayer spacing, and porosity. Su et al. prepared high-quality graphene sheets by electrochemical exfoliation of graphite. The electrochemical cell consists of graphite anode and platinum cathode in a 0.5M of HMorales et al. used an electrochemical route to produce few-layer graphene from electrochemically-produced graphite intercalation compounds in aqueous perchloric acid. Anodic intercalation is more efficient in terms of time. To prevent the formation of graphite oxide, cathodic pretreatment is preferred. Experimentally, it was found that using electrochemical-potential control is very promising to obtain graphene with different degrees of oxidation [151].An electrochemical route for producing graphene sheets by electrochemical intercalated sodium dodecyl sulfate (SDS) into graphite followed by electrochemical exfoliation of an SDS-intercalated graphite electrode was reported by Alıoğlu et al. [152]. The structural order, size, and a number of layers of synthesized graphene sheets depend on the potential value for SDS intercalation into graphite. Graphene sheets with the highest structural order and lowest number of layers can be obtained using relatively high intercalation potentials. The average size of 500 nm and a monolayer of graphene sheets were prepared at high SDS intercalation potentials into graphite. After successive electrochemical intercalation and exfoliation processes, UV–vis spectra of graphene/SDS suspensions showed a large amount of the reduced form of graphene flakes was obtained [152].4)2SO4 aqueous solution was reported by Chen and Xue [153]. The proposed electrochemical exfoliation mechanism showed that SO42− and H2O could be intercalated into those graphite sheets. Monolayer and few-layer graphene were obtained due to the SO2 and O2 gases formed within graphite sheets. The exfoliated colloidal graphene can be perfectly stabilized in DMF solvent for more than 1 week. The colloidal graphene can show promising applications in flexible printed electronics and energy storage devices [153]. Anodic exfoliation of graphite in  and a graphite rod were employed in the 3-electrode system, consisting of a platinum wire counter electrode and silver wire (with glass frit) or platinum wire as the pseudo- reference electrode. Highly orientated pyrolytic graphite (HOPG) gives greater advantages in terms of the exfoliate size, but there are difficulties in the electrode set up and require the use of sonication. The use of graphite rod electrode does not require ultrasonication or any secondary physical processing of the resulting dispersion. Few-layer graphene flakes with 2 nm thickness are formed directly with flake diameters from 100–200 nm [154]. 4)2SO4, followed by sonication to separate the oxidized graphene sheets, and then reduce oxidized graphene to graphene. With this strategy, it is possible to convert around 30 wt % of the initial graphite into graphene with short processing times and obtain high yields. The graphene sheets are well dispersed in water, have a lateral size of about 0.5–1 μm, and contain only a few graphene layers, most of which are bilayer sheets [155]. Seville et al. [155] presented a facile and environmentally friendly synthetic strategy to produce stable and easily processable Graphene dispersions in water. This method is an alternative to classical chemical exfoliation methods such as the Hummers method, which is more complex, dangerous, and harmful. The new strategy is based on the electrochemical exfoliation of graphite. It includes three simple steps: anodic exfoliation of graphite in . Using different intercalation potential values of 1, 3, 5, and 7 V, the SDS was intercalated into graphite anode electrode. Then, the SDS-intercalated graphite electrode was exfoliated in the same medium by acting as a cathode. Stable graphene dispersions were obtained after these two electrochemical steps. A new glassy carbon electrode (GCE) was modified with graphite, graphene/GCE. This new graphene/GCE electrode has good electrocatalytic activity and has been used for amperometric determination of nitrite in both standard laboratory and real samples [156]. 2SO4, and a high voltage DC is applied, which exfoliates graphite into graphene. Thus, graphene is collected and then oxidized into GO by chemical oxidation using MHM [157]. The process is easy and manageable for large-scale production. The quality of GO prepared can be controlled by sweeping the voltage and type of electrolyte [97].The synthesis of GO by the electrochemical method is utilized recently. This involves the electrochemical conversion of a graphite electrode (as an anode) into graphene under high potential, followed by its chemical oxidation. The graphite anode is inserted in the electrolyte solution, i.e. H2SO4 [158], and aqueous solutions of different inorganic salts ((NH4)2SO4, Na2SO4, K2SO4, etc.) [159]. A high yield of FLG (>70%) was prepared by electrochemical charging of graphite electrodes in a Li+/propylene carbonate electrolyte. This high yield of FLG obtained from the graphite electrode is significantly higher than that produced by most current liquid-phase exfoliation methods [160]. Generally, the electrolyte for this process can be ionic liquids [126], aqueous acids such as HThe electrolyte plays a crucially important role in the yield and quality of electrochemically synthesized Graphene. A lower amount of graphene is produced by exfoliation in ionic liquids with a small graphene’s small lateral size. The electronic properties of the produced graphene are distorted due to functionalization with ionic liquids [126]. However, high-quality graphene with a large lateral size is produced via exfoliation in acidic electrolytes. Nevertheless, over-oxidation of graphite by acidic electrolytes leading to the presence of oxygen-containing functional groups .2O2/H2O) system that can produce high-quality, anodic few-layer graphene nanosheets in 95% yield at ambient reaction conditions [162].A facile, mild, and environmentally-friendly approach for the efficient electrochemical exfoliation of graphite was developed using a sodium hydroxide/hydrogen peroxide/water (NaOH/HExfoliated graphene oxide/multilayer-graphene (GO-MF) flakes were obtained by electrochemical exfoliation of industrial graphite using tungsten (99.5% W) as the counter electrode and graphite as the working electrode [163]. The analytical techniques and XRD results showed 67 wt.% graphene oxide (GO) and 33 wt.% multilayer-graphene (MG). The mechanism of electrochemical exfoliation of graphite in ionic liquid solution involves (i) anodic oxidation of water, (ii) hydroxyl and oxygen radicals’ generation, which attacks the edges of the various graphite sheets (iii) intercalation by ionic liquid anions between graphite layers, forming the graphite-intercalation-compounds (GICs), and (iv) oxidative cleavage and precipitation of the GICs [164]. Lin et al. showed the electrochemically exfoliated graphene flakes have higher crystallinity and fewer defects than graphene flakes prepared by liquid phase exfoliation [165].2SO4. The size of SO42- ion in solution is ~4.60 Å, which is about ~1.37 times higher than the interlayer spacing of 3.35 Å between graphitic layers in graphite. These SO42- ions under applied voltage intercalate between graphitic layers, and they push the graphitic layers farther apart, making the spacing greater than 3.35 Å. This leads to graphite exfoliation because of the weakening of Van der Waals bonding .Most of the graphene synthesis through electrochemical exfoliation has used acidic electrolytes typified by HIn electrochemical exfoliation with alkaline electrolyte, both OH ions (have a size of ~0.958Å) and the hydrated OH ions (have a size of ~2.503 Å) are smaller than the graphitic interlayer spacing of 3.35 Å. The electrolyte with hydrated OH- ions (size ~2.503 Å) behave as polar, and due to the applied electric field, they enter the graphitic layers. These ions will polarize the graphitic layers located above and below, and the hydrated OH- ions will get coupled with the graphitic layers with electrostatic interaction. Under optimum electric force, the hydrated ions will pull the graphitic layers above and below out of the graphitic stack. The graphene-hydrated OH- configuration forming bilayer Graphene will thus get formed and float in the alkaline electrolytic solution [166]. The three main advantages of an electrochemical method of graphene production are: Firstly, it is used to obtain high-quality graphene with no use of hazardous oxidizing materials. Secondly, the method is relatively simple such that a scale-up to industrial production levels could be comparatively straightforward. Moreover, thirdly, hazardous chemicals and the lack of necessity for temperature control and makes the electrochemical production of GO more environmentally friendly . Table 1 summarizes the previous and recent representative studies on the synthesis of graphene by exfoliation from different carbon sources and reacting media.This paper reviewed the most common graphene fabrication methods. It involves the concept and technical development and modification in each process since its first inception in 1898. This review focuses on graphite exfoliation and its progress because it is considered the most popular method for graphene production with high yield and different properties. Most of the scientific works are interested in the mechanism of exfoliation, targeting how to get more yield, low O content, fewer layers, and large lateral size, best properties, low cost, more eco-friendly, and with fewer steps. Many excellent reviews have been published on the production and processing of graphene, including Cai et al. [13], Bonaccorso et al. [119], and Chung et al. [104].Cai et al. [13] presented an excellent general review on graphite exfoliation methods and discussed the bulk production of graphene via exfoliation, focusing on the exfoliation techniques and yields. Different precursors are used for exfoliation such as graphite oxide, pristine graphite, graphite intercalation compounds, and expanded graphite, but the yield of monolayer graphene is still relatively low. Furthermore, another drawback to the exfoliation of graphene sheets in solvents is the aggregation of these sheets due to Van der Waals attraction, which compromises the exfoliation efforts. Chung et al. [104] gave an excellent general review on exfoliated graphite (EG), which is graphite that has a degree of separation of a substantial portion of the carbon layers in the graphite. Graphite nanoplatelet (GNP) is commonly prepared by mechanical agitation of EG. Bonaccorso et al. [119] reviewed graphene as a new class of 2D materials derived from layered bulk crystals. They emphasize the requirement of graphene application and the development of industrial-scale, reliable, inexpensive production processes.


Current limitations of cell scaffolds include the lack of electrical conductivity and suboptimal mechanical properties. Here we discuss how the incorporation of graphene into cellular scaffolds, either alone or in combination with other materials, can affect morphology, function, and maturation of cardiac cells. We conclude that graphene-based scaffolds hold great promise for cardiac tissue engineering.Cardiac tissue engineering requires materials that can faithfully recapitulate and support the native  Cardiomyocytes are electrically and mechanically active cells. Therefore, to efficiently support their activities, the cardiac substrates/scaffolds must have matching properties in order to faithfully recapitulate the functional behavior of the myocardium. Despite technological advances, this task remains very challenging, and new materials that can address these problems are in high demand.Graphene, the newest member of the carbon allotrope family, is an exceptional candidate for this role. Although other members of this family are already famous , graphene could outshine them all. Graphene, a two-dimensional (2D) carbon crystal, is often described using superlatives: graphene is the thinnest (0.335 nm), the lightest (0.77 mg/m2), and the strongest (42 N/m) material . ElectriGraphene came into the spotlight in 2010 with the Nobel Prize in Physics awarded “for groundbreaking experiments regarding the two-dimensional material graphene” . It beca2-hybridized, and each atom allocates 3 electrons from its 4 outer shell electrons to form equivalent covalent σ-bonds with its three neighboring atoms. The 4th electron which occupies a pz orbital is forming a π bond. Unlike the sp2 orbitals, the pz orbitals do not directly overlap, and these 4th electrons (π electrons) operate in the 3rd dimension (above and below a 2D graphene sheet), allowing them to be highly mobile, behave as massless particles, and experience ballistic transport without scattering , and the graphene’s current density is ∼ 1,000,000 times greater than in copper  and large Youngs modulus (∼1 TPa) (Strong carbon-carbon in-plane σ-bonds (the bonding energy of 4.93 eV)  make gra(∼1 TPa) . Defect-−1 and high mobility of 320 cm2 V−1 s−1 (Graphene oxide (GO) is an oxidized form of graphene containing oxygen functional groups, the presence of which, among other consequences, leads to the disruption of the sp2 structure, increased interlayer spacing, and low electrical conductivity. Reduced graphene oxide (rGO) is a deoxygenized form of GO. Although the graphene structure in rGO is not completely restored by the reduction processes due to some remaining oxygen groups and surface defects, removal of oxygen groups and the increased C/O ratio leads to restoration of the sp2 structure, increased mechanical strength, surface area and stability, hydrophobic properties, and the increase of electric conductivity up to 6300 S cm V−1 s−1 .E. Coli , surface functionalization (intended and unintended), and dosage are the factors affecting how graphene may interact with cells . After bCurrently, the scientific community appears to be in agreement that properly engineered graphene materials are not only biocompatible but often superior in providing a microenvironment necessary for cell growth, differentiation, and development .To evaluate the graphene effects on cell stress, several studies monitored such highly sensitive cell viability indicators as autophagy levels and mitochondrial morphology and membrane potential . These sin vitro models of cardiac syncytium -derived cardiomyocytes, and human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes) cultured on graphene-based planar substrates fabricated from rGO or chemical-vapor-deposition (CVD) graphene. Further, zebrafish embryos injected with graphene flakes (5–20 μm in diameter) were exhibiting the basal heart rates similar to control embryos, and all but one zebrafish retained full viability 3 days later, which confirms biocompatibility of graphene in vivo , muscle contraction and relaxation (troponin-T), and cytoskeleton alignment (actinin-4) even without electrical stimulation .Cardiomyocytes on a vitronectin-coated graphene substrate exhibited enhanced expression of cardiomyogenic markers and cardiac-specific extracellular matrix genes even without addition of cardiomyogenic factors. These results suggest that, by promoting the absorption and correct presentation of vitronectin, graphene induces mesodermal and endodermal lineage signaling which supports the cardiac development .Another study transferred graphene films onto polymeric topographic substrates to create a conductive nanopatterned anisotropic scaffold. Cardiomyocytes on this scaffold demonstrated more mature structural and functional properties. These cardiomyocytes exhibited significantly increased z-band widths, greater sarcomere lengths, and increased expression of Cx43. Additionally, the amplitude of calcium transients and the action potential duration were significantly greater, suggesting enhanced maturation of cardiomyocytes .in vivo are inherently 3D, a similar geometric arrangement of engineered heart tissues (EHTs) would be more desirable to ensure faithful recapitulation of cell-to-cell coupling, and complex organization and function. Incorporation of graphene into cardiac 3D scaffolds can lead to anisotropic nanotopology, greatly enhanced electroconductive properties, and provide the ability to perform cell stimulation and monitoring via cell scaffolds.Since cardiac tissues A graphene foam  itself cAnother option is to directly embed graphene materials into 3D preparations. This approach was shown to increase the number of successfully differentiated 3D embryoid bodies and improve the mechanical and electrical properties of differentiated cardiomyocytes . FurtherPark et al. combined GO flakes (1–6 μm in diameter) with MSCs and discovered that it led to more efficient engrafting of cells at the ischemic lesion site . It appeα-actinin, and supported strong spontaneous synchronous contraction (20–30 BPM).Alternatively, cardiomyocytes and graphene materials can be combined to fabricate a complex multilayer cell construct. Shin et al. deposited a poly-L-lysine-GO nanofilm layer onto a homogenous layer of cardiomyocytes, seeded another layer of cells on top of the graphene-enhanced nanofilms, and then repeated these steps until the constructs comprised of 3 or more cell layers . This scGraphene-containing 3D scaffolds can also be fabricated by electrospinning to develop the scaffolds with enhanced mechanical and electrical properties . employedα-MHC)  .In another development, poly (caprolactone) (PCL)/poly (glycerol sebacate) (PGS) nanofibers were fabricated via electrospinning, and graphene  was added to PCL/PGS nanofibers. Addition of graphene to PCL/PGS nanofibrous scaffolds led to improved electrical conductivity, balanced hydrophilicity, and increased surface roughness. Adhesion, growth, migration, proliferation, and viability of cultured human cardiomyocytes were increased with the increase of the ratio of graphene in PCL/PGS scaffolds .Talebi et al. used electrospinning technique to fabricate highly hydrophilic fibrous scaffolds composed of polycaprolactone/chitosan/polypyrrole (PCP) and graphene . PCP-gravia electrospinning. When used for cardiac differentiation of human iPSCs transfected with TBX18 gene, rGO-SF scaffolds exhibited improved mechanical and electrical properties, acceptable biocompatibility, considerable cell attachment, enhanced maturity and upregulation of cardiac genes   scaffolds, rGO nanosheets were incorporated into SF nanofibers d α-MHC) . Alternad α-MHC) .The incorporation of graphene materials into hydrogels results in hybrid electroactive scaffolds with improved cell adhesion, desirable mechanical properties, and low immunogenicity.Actn and cTnT) improved; 3) cardiomyocytes reached synchronized contractions within 48 h, contracted in a more uniaxial manner and with increased amplitude, and continued contracting for up to 3 months  more cardiomyocytes were binucleated and had longer sarcomere lengths much sooner; 2) the cardiac gene expression  hydrogel, which leads to significant enhancement of electrical and mechanical properties of the hybrid material . NeonataAnother study utilized GelMA hydrogels with incorporated carbon nanotubes, GO, or rGO to compare the effects of these materials on the structural organization and functionality of hiPSC-cardiomyocytes . It was To engineer advanced EHTs, Tsui et al. used a decellularized extracellular matrix harvested from the left ventricular myocardium of porcine hearts to create a hydrogel composite scaffold with a preserved tissue-specific protein profile and a tunable stiffness enhanced by rGO . MechaniJing et al. developed polydopamine-based chitosan/GO composite hydrogels and demonstrated that addition of GO increased the hydrogel’s adhesion strength by 300%, and improved its electrical conductivity . FurtherMyocardial infarction causes irreversible damage to myocardium and results in an increased risk of heart failure and sudden cardiac death. One strategy to address this problem is to integrate new MSCs or cardiomyocytes into the remaining myocardium. Graphene-containing injectable hydrogels could improve the efficiency of engraftment and enhance the survival of implanted cells.TrpT-2, Cx43, and Actn4 even without electrical stimulation.An injectable rGO-enhanced alginate hydrogel was used for culturing MSCs , resultiAnother study combined methacryloyl-substituted tropoelastin with GO nanoparticles to engineer an injectable, stretchable, conductive hydrogel with an enhanced elasticity and toughness . This GOTo address the loss of vasculature at the ischemic site, Paul et al. employed an injectable graphene-enhanced hydrogel composed of a low-modulus methacrylated gelatin wtih polyethylenimine functionalized GO nanosheets to deliver a proangiogenic vascular endothelial growth factor plasmid DNA to the damaged myocardium . In a raZhao et al. developed an injectable Reverse Thermal Gel (RTG) functionalized with GOs (GO–RTG) and capable of forming a 3D matrix at 37°C . Using nin vivo applications. It is important to note that the graphene research is still in its infancy, and new fundamental discoveries and new practical applications are emerging every day, further fueling the development of the next wave of biomedical applications. We wholeheartedly share the sentiment expressed by scientists from the University of Manchester, a home of 2010 Nobel Prize Laureates for the research on graphene that “the potential of graphene is limited only by our imagination”.Numerous reports provided compelling evidence that graphene can play an essential role in creating a more physiologically accurate microenvironment for cardiac cells and EHTs. However, more research needs to be done to optimize the composition and geometry of graphene-based scaffolds for complex long-term


GAFs show high anisotropy on average cross-plane thermal conductivity (K⊥) and average in-plane thermal conductivity (K||). Additionally, uniaxially compressed GAFs performed a large elongation of 11.76% due to the Z-shape folding of graphene layers. Our results reveal the ultralight, ultraflexible, highly thermally conductive, anisotropy GAFs, as well as the fundamental evolution of macroscopic assembled graphene materials at elevated temperature.Graphene aerogels have attracted much attention as a promising material for various applications. The unusually high intrinsic thermal conductivity of individual graphene sheets makes an obvious contrast with the thermal insulating performance of assembled 3D graphene materials. We report the preparation of anisotropy 3D graphene aerogel films (GAFs) made from tightly packed graphene films using a thermal expansion method. GAFs with different thicknesses and an ultimate low density of 4.19 mg cm The 3D nt years ,5. Compant years ,7,8,9. Gnt years . The print years ,12,13. Mnt years , polymernt years .−1 K−1 at room temperature [−3 to 1.7 W m−1 K−1 [The thermal conductivity of single-layered graphene measured by the noncontact Raman spectroscopy technique is in the range of~3000–5300 W mperature . It showperature . To explperature . Using t m−1 K−1 ,19,20,21−3. We studied the impact of thermal treatment on the structural change of graphene sheets and the evolution of their packing situation. We proposed a mechanism for forming 3D GAF architectures relating to the interlayer accumulation and expansion due to the decomposed gas. The GAFs show highly anisotropic conductive properties. The cross-plane thermal conductivities (K⊥) were only 0.3–0.7 W m−1 K−1 due to the relatively low densities. The K|| value showed a rapid increase from 8.98 to 53.56 W m−1 K−1 along the in-plane direction, when the density increased from 40.32 mg cm−3 to 150.49 mg cm−3. Besides, compressed GAFs with a thickness of 19 µm showed an ultrahigh elongation of 11.76% during the tensile test. This high-temperature thermal expansion is convenient access to the unique oriented 3D architectures of GAFs and attracts anisotropic thermal performances.The present study demonstrates ultralight, ultraflexible, anisotropic and highly thermally conductive graphene aerogel films (GAFs). Utilizing the opposite process of sheet-assembly, we employed a thermal expansion method starting from tightly packed graphene films. The resulting GAFs still kept the film appearance in the macroscopic view, forming a microscopic 3D porous network, reaching an ultralight weight of 4.19 mg cmGraphene oxide (GO) was purchased from Hangzhou Gaoxi Technology Co. Ltd. . Other chemical reagents were all analytical grade and used as received. GO dispersion was prepared using deionized water (18.2 MΏ cm).GO films were prepared by vacuum filtration of graphene dispersion. The thickness could be controlled easily by adjusting the amount of GO dispersion. The GO film was fixed on a Teflon plate, soaked in 40% of the HI solution, and reduced to 12 h at 80 °C. Then the RGO film was rinsed with water, saturated sodium bicarbonate solution, water and ethanol solution, and dried in a vacuum of 100 °C for 12 h to obtain the reduced graphene film.Heat treatment was carried out using a high-temperature graphitized furnace, and the RGO is placed in a graphite box, put into the medium frequency high-temperature graphite chemical furnace under the protection of argon heating treatment, heating up to the target temperature and kept for a time . GAFs were compressed using vertical oil pressure jacks that hold for 1 h at 50 MPa.−1 was applied in all mechanical tests. SEM images of the fracture section determined the cross-section area. Thermal diffusivity was measured by a Netzsch LFA 457Gerätebau GmbH, Wittelsbacherstr.laser flash thermal diffusivity apparatus. The thermal conductivity of the GAFs was determined according to Ref. [SEM images were obtained using a Hitachi S4800 field emission SEM system. XRD patterns were collected on a Philips X’Pert PRO diffractometer equipped with Cu Kα radiation  with an X-ray wavelength (λ) of 1.5418 Å. Raman spectra were taken on a Renishaw in Via-Reflex Raman microscopy at an excitation wavelength of 532 nm with less than 1 mW laser power and 10 s integration time. The tensile tests were carried out on an HS-3002C mechanical testing system. GO, and graphene films were cut to 3 mm wide films. Both ends of the individual film were fixed onto clamps made by two PDMS slices with gauge lengths of 10 mm, respectively. A loading rate of 2 mm min to Ref. .The thickness of the annealed graphene film increased with the processing temperature. The cross-sections of graphene films annealed at various temperatures are presented in −1 for GO and HI-reduced GO, respectively, just as it did for the monolayer graphene but with a larger linewidth [−1 with the increasing annealing temperature, indicating the graphic structure’s effective recovery. The 3000 °C treatment extensively repaired the conjugated system of graphene and resulted in defect-free graphene [The annealing process on graphene films was followed by Raman spectroscopy . The ID/inewidth . The wavgraphene . The evoX-ray diffraction (XRD) patterns show that the 2θ peak values of graphene films increase with the annealing temperature. Meanwhile, the corresponding d-spacing gradually approaches the value of graphite. Detailed information for GAFs annealed from 500 °C to 3000 °C are obtained in −3), while it still kept the film appearance in macroscopic view. Besides, resulted GAFs showed an ultimate ultralight weight of 4.19 mg cm−3. Vertical expansion results in diameter shrinkage, because the conformation of sequential horizontal stacked graphene layers transformed into deformation along the in-plane direction. Observed from the SEM images of a film cross-section was made by tear opening, the GAFs burst into a loose and porous structure , which are highly flexible and easy to stack. GAFs with different thicknesses were compressed into compact films using the method for making graphite devices. The compression was carried out using a plate vulcanizing machine under 100 MPa pressure. Compressed GAFs showed high flexibility . Even whCompressed GAFs are very different from traditional graphite films made by compression molding. Firstly, the compressed graphene films are smooth and uniform with a silvery metallic luster, while graphite films are black. Secondly, the graphene film is very flexible, while the graphite film is fragile with insufficient flexibility. Thirdly, in the microcosmic view, compared with the unoriented graphite crystalline domain in graphite films, graphene layers in GAFs are highly oriented in the vertical direction, which is more evident in thicker ones . The lay−1 K−1. Practically, macroscopic graphene aerogels are usually thermally insulating because of the high porosity and low air cross-convection. Statistically, graphene sheets in normal 3D monoliths are oriented randomly, which results in isotropic thermal diffusivity in macroscopic graphene aerogels. In this study, high-temperature thermal annealing and expanding results in an anisotropic structure and thermal transport properties. The final GAFs formed a structure in which graphene layers are continuously paralleled in the vertical direction and enclose many air chambers. The incorporation of lateral orientation and high structural integrity contributes to the enhancement of phonon transmission in the lateral plane for thermal transport.Single-layered graphene has an extremely high thermal conductivity of 5300 W m|| and α⊥) for a GAF with a thickness of 1 mm and a compressed graphite plate from 25 °C to 500 °C are shown in 2 s−1 (α||) and 421.5 mm2 s−1 (α||) for GAF at 25 °C, while 23.5 mm2 s−1 (α⊥) and 106.3 mm2 s−1 (α||) for GAF at 25 °C. The anisotropy (α⊥/α||) decreased monotonically from 5.61 at 25 °C to 4.52 at 500 °C. In contrast, the compressed graphite plate showed slightly higher α|| than α⊥, and, thus, very low anisotropy α⊥/α|| from 1.29 to 1.07. This result indicates that the GAFs possess a highly aligned structure and exhibit much higher anisotropy than the isotropic compressed graphite plate.The anisotropy of thermal conductivity was characterized using the laser flash method. Among non-steady-state methods, laser-flash thermal diffusivity measurement is widely used for its low limitation on sample size, rapid and stable measurement, simple calculation process, adjustable laser pulse energy and width, strong applicability, and wide measurement temperature range. The temperature-dependent in-plane and cross-plane thermal diffusivities (α⊥) fluctuated at 60–120 mm2 s−1 without evident tendency till the thickness of GAFs decreased below the detective limit (⊥) were only 0.3–0.7 W m−1K−1 due to the low densities of 4~10 mg cm−3. On the other hand, the α|| value rapidly increased from 293.46 to 448.23 mm2s−1, and the corresponding thermal conductivities (K||) were from 8.98 to 53.56 W m−1K−1 with the density increasing from 40.32 to 150.49 mg cm-3 (||/K⊥) was 29.93 for GAF of 40.32 mg cm−3 and 76.51 for GAF of 150.49 mg cm-3. Final compressed GAFs had a thermal conductivity of 734 W m−1 K−1 at a density of 1.59 g cm−3. Besides, the electrical conductivity of the compressed GAFs was ~105 S m−1, much higher than that of HI-reduced graphene films [The density-dependent thermal performance for GAFs was further studied using a stepwise uniaxial compression. The cross-plane thermal diffusivities  ranging from 29.93 to 76.51, and an ultrahigh elongation of 11.76% for 19 µm-thick graphene films after uni-axial compression. The ultralight, ultraflexible and highly thermally conductive anisotropy of GAFs should lend them to many applications in thermal interfacial conducting, electromagnetic shielding and energy storage.In summary, 3D graphene aerogel films (GAFs) prepared from tightly packed graphene films were successfully developed by a facial high-temperature thermal expansion method. The isolation, accumulation, and expansion of decomposed gases combined with relaxation, slippage, and buckling of graphene sheets at 3000 °C gave a loose and porous structure to the GAFs. The 3D graphene film architecture exhibits an ultralight weight of 4.19 mg cm


The impact of M(IL4) treatment on C. rodentium infectivity, colon histopathology, tumor number and size and tissue-specific inflammation was examined in these models. The anti-colitic effect of the M(IL4)s were confirmed in the di-nitrobenzene sulphonic acid model of colitis and the lumen-to-blood movement of 4kDa FITC-dextran and bacterial translocation to the spleen and liver was also improved by M(IL4) treatment. Analysis of the other models of disease, that represent comorbidities that can occur in human inflammatory bowel disease (IBD), revealed that M(IL4) treatment did not exaggerate the severity of any of the conditions. Rather, there was reduction in the size (but not number) of polyps in the colon of AOM/DSS-mice and reduced infectivity and inflammation in C. rodentium-infected mice in M(IL4)-treated mice. Thus, while any new therapy can have unforeseen side effects, our data confirm and extend the anti-colitic capacity of murine M(IL4)s and indicate that systemic delivery of one million M(IL4)s did not exaggerate disease in models of colonic or airways inflammation or colonic tumorigenesis.The murine interleukin-4 treated macrophage (MIL4) exerts anti-inflammatory and pro-healing effects and has been shown to reduce the severity of chemical-induced colitis. Positing M(IL4) transfer as an anti-inflammatory therapy, the possibility of side-effects must be considered. Consequently, bone marrow-derived M(IL4)s were administered  Typically associated with the phagocytosis of microbes, macrophages perform a myriad of functions in host defense and homeostasis. This versatile cell is highly responsive to its’ microenvironment and has been broadly classified into : classicin vitro differentiated murine M(IL4)s significantly reduced the severity of dinitrobenzene sulphonic acid (DNBS), oxazolone, and dextran sodium sulphate (DSS) induced colitis in mice: in contrast, the administration of CAMs differentiated in vitro with IFNγ [or control M(0)] did not impact the outcome of the colitic disease s as an anti-colitic therapy are available; however, with any new therapy, there is the possibility of side-effects. For instance, would the M(IL4)s pro-healing effect, if uncontrolled, result in fibrosis? While not identical, the M(IL4) and other AAMs share some similarities with myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs), raising the possibility that M(IL4)s could promote or exaggerate tumorigenesis . It has Citrobacter rodentium-infected mice was reduced by M(IL4) co-treatment. These proof-of-principle findings lend support for M(IL4)s as an anti-colitic therapy, and if translatable to human, suggest that M(IL4) treatment side-effects may be limited and would not offset the therapeutic benefit of this cellular immunotherapy.Cognizant of these possibilities, the current study was designed to determine if murine M(IL4)s would affect the severity of disease in models of inflammation-associated colon cancer (CRC), infectious colitis and antigen-driven airways hypersensitivity. All of these conditions can be comorbidities in human inflammatory bowel disease (IBD), the target condition for M(IL4) treatment. The data herein, confirm and extend the anti-colitic effect of intraperitoneal delivery of M(IL4)s, and reveal that these cells, under the paradigms tested, did not boost tumor development in the azoxymethane (AOM)/DSS model of CRC, or immune cell infiltration and lung histopathology in the ovalbumin model of airway hypersensitivity. Intriguingly, inflammation in Animal experiments were approved by the University of Calgary Animal Care Committee in compliance with the guidelines of the Canadian Council on Animal Care and administered under protocol AC17-0115.Following a published protocol , bone ma6 in 500 μL PBS), 48h before intra-rectal instillation of DNBS   received an intraperitoneal (ip.) injection of M(IL4)s    could p+, FIZZ1+, Ym1+). Treatment with agents that block AAM activity can reduce the severity of disease in models of airway hypersensitivity/asthma (+ AAMs can be anti-fibrotic (+/FIZZ1+ AAM obtained from antibiotic-treated mice increased inflammation in the airway (in vitro differentiated M(IL4)s given prior to allergen challenge had no effect in an OVA-model of airways hypersensitivity. M(IL4)-treated mice did not display an increase in collagen indicating no propensity towards fibrosis in this acute model setting. A variety of chemical and allergen models of airways inflammation are available, and it will be important to determine if M(IL4)s affect the inflammation, fibrosis or lung function in these models s and the preservation of gut barrier function, the current study yielded no data to suggest that M(IL4) therapy would exaggerate or leave an individual more vulnerable to concomitant infection, CRC or airways inflammation. These observational data are presented in support of M(IL4) cellular therapy and could be augmented by mechanistic studies to elucidate, for example, how the administration of M(IL4)s resulted in reduced polyp size in the AOM/DSS model of CRC. We recognize that the range of potential side effects of any new therapy is large and we have considered but three of these, and that further testing of M(IL4)s in variations of the models presented herein and other disease model systems is important to garner a more holistic view of the M(IL4) in disease. Given that sex-differences have been noted in disease, it will be important to broaden the assessment of putative M(IL4) side effects by performing experiments with female mice  in models of disease. Thus, continued research efforts are required to precisely elucidate the anti-colitic mechanism of M(IL4)s and to test M(IL4)s in other models of infection, cancer and inflammatory disease (e.g. cirrhosis) , and to The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by University Calgary Animal Care Committee in compliance with the guidelines of the Canadian Council on Animal Care and administered under protocol AC17-0115.BC, project design, data collection and analysis, manuscript writing & review. GB, data collection and analysis, manuscript review. NJ, data collection and analysis, manuscript review. AW, data collection and analysis, manuscript review. AB, data collection and analysis, manuscript review. KK, blinded tissue scoring, analysis and manuscript review. RW, manuscript review. MK, blinded tissue scoring, analysis and manuscript review. EC, manuscript review. DM, project design, grant support, analysis, manuscript writing and review. All authors contributed to the article and approved the submitted version.This work was supported by a Grant-in-Aid from Crohn’s Colitis Canada to DM. BC is supported by an Alberta Innovates-Health Sciences Post-doctoral Fellowship. NJ is supported by the Francis Family Foundation as a Parker B Francis Fellow. Work from the Cobo laboratory is supported by a Discovery Grant from the Natural Sciences and Engineering Council of Canada (NSERC), RGPAS-2017-507827. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.