A test method for evaluating the quality of graphene flakes, such as reduced graphene oxide (rGO) and graphene nanopowder (GNP), was developed in this study. The pelletizer was selected for a sampling tool, which enables us to formulate the flake sample as a measurable sample. Various parameters were measured from the pelletized sample in order to elucidate the best parameter for representing the quality of the graphene flakes in terms of their electrical properties. Based on the analysis of 4-probe measurement data on the pelletized sample, the best intrinsic parameter is volume resistivity (or volume conductivity) rather than resistivity (or conductivity). Additionally, the possible modification of a sample before and after the pressurization was investigated by electron microscopy and Raman spectroscopy. No significant modification was observed. The volume conductivity in the two types of the graphene was different from their individual conductivities by one order of magnitude. Based on the results of X-ray photoelectron spectroscopy and Raman spectroscopy measurements, the volume conductivity of the graphene flake samples was governed by the oxygen content in the sample. Our achievements will promote the effective use of powder-type graphene products for further applications. To date, two types of graphene, namely, films and powders have been developed for specific applications. The film-type graphene fabricated by the chemical vapour deposition (CVD) or epitaxy methods has been studied for replacing the indium-tin oxide (ITO) glass that is widely used in the current display industry5. Furthermore, graphene films can be used to fabricate flexible transparent conducting films that can be used as flexible substrates in printed electronics or wearable electronics7. The powder-type graphene that is mainly produced by reduction of graphite oxide monolayer is the other type of graphene that may enable more practical applications, for example serving as an energy material with a high density as well as an outstanding electrical conductivity10. The use of powder-type graphene can be extended to the formation of functional composites due to its lower density and high mechanical properties12.Graphene has attracted much attention due to its extremely high mobility and ballistic transport of electrons, which enable its utilization as a next-generation electronic material14. Recently, more effort has been made in utilizing the powder-type graphene in industrial applications due to another advantage of this novel material. Since powder-type graphene exhibits greater versatility for use with other matrices, it can be a good candidate for use as an anodic material for secondary batteries, as an additive to activated carbon for supercapacitors, and in other applications16. In particular, the introduction of chemical exfoliation for the fabrication of powder-type graphene can reduce its production cost and enhance large scale productivity, possibly accelerating the use of powder-type graphene product in industrial applications in the near future19.During the first decade after the discovery of graphene, most work in academic and industrial research has been done on the film-type graphene. However, technical obstacles must still be overcome to enable the economically viable use of film-type graphene as a transparent conducting electrode in display industry because currently, homogeneous electrical conductivity cannot be obtained over the entire surface20. As the nature of powder-type graphene is arguable, it is essential to define the term and evaluate the material. Fortunately, the discussion on these issues have been actively made by several technical groups, especially by standardization societies such as ISO  and IEC . In 2017, ISO published the technical specification (TS) which defines graphene and classified the graphene in terms of geometric factors21. In this TS graphene is categorized by four types such as graphene, bilayer graphene, few-layer graphene, and graphene nanoplatelet. The controversy on the definition of graphene nanoplatelet is forced to use graphene flake20, which is widely used, instead of powder-type graphene here-in-after in this paper to our best knowledge.Since powder-type graphene is expected to be widely used as mentioned above, there is high demand from both the manufacturers and users for developing evaluation methods that can be applied to the powder-type graphene products22, it is necessary to select the parameter that best represents the electrical quality of graphene flakes. Graphene flakes are generally produced by the exfoliation of graphite in an acidic solution18. In its manufacturing process, a layer of graphene is fabricated via oxidation. Hence, a reduction process should be used after the oxidation process to produce a powder product which is the so-called reduced graphene oxide (rGO). Consequently, the electrical properties of such graphene are influenced by its oxygen amount and flake size24. Therefore, it is necessary to identify suitable sample preparation and measurement conditions for obtaining the target properties.Since the electrical properties of graphene flakes are simultaneously correlated to its geometric and electronic characteristics26. To measure the electrical properties, it is necessary for the powder sample to be formulated as a pellet under pressurization. As the applied pressure is varied, two parameters such as the resistance and thickness of the pellet are directly measured at the given pressure28. The sheet resistance is calculated by considering an instrument-dependent correction factor (F)30. From the sheet resistance data, the resistivity of the sample is calculated by taking the obtained thickness value into account. Since the resistivity or the conductivity of a sample is an intrinsic property, it should be equal or close to that of the CVD–grown graphene. Another candidate parameter to should be considered is volume resistivity or volume conductivity which is a function of the given pressure26. Accordingly, we selected the following parameters to determine how well they represent the quality of graphene flakes in this study: (i) sheet resistance (or conductance), (ii) resistivity (or conductivity), and (iii) volume resistivity (or volume conductivity). Based on a systematic review of the results for these three parameters obtained for different types of graphene samples, the best parameter for the characterization was determined.Generally, the powder resistivity measurement method is used to investigate the electrical properties of the powder samplesIn addition to electrical characterization of the flakes under pressure, the possible modifications of the sample structure before and after the pressurization were examined by electron microscopy and Raman spectroscopy. The quantitative and qualitative analysis of carbon and oxygen, which are the main components in the samples, was also performed using X-ray photoelectron spectroscopy (XPS). These results will be used for evaluating the electrical quality of the graphene products and for determining the key characterization parameters in order to extend their applications.31.Three types of graphene flakes were used in this study, namely, i) two commercialized brands labelled as rGO by their manufacturers and ii) graphene nanopowder (GNP) which is also commercially available. Since the number of layers, which is indicated by the thickness, is a key parameter of a graphene product, the thickness data provided in the material specifications of these products were confirmed by transmission electron microscopy (TEM). High-resolution TEM images and selected area electron diffraction (SAED) patterns of the as-received graphene materials are shown in Figs The morphology of the three samples shown in Fig. −1 and disorder modes (D-band) at approximately 1340 cm−132. Since the D/G ratio (ID/IG) is generally considered to be a measure of the defect density of carbon materials containing sp2 bonding33, possible structural deformations before and after pressurization were examined through the changes in the D/G ratios. A significant variation in the ratio was not observed in the three types of graphene flakes , pellet thickness (t), pellet density (dv), sheet resistance (ρs), resistivity (ρ) and conductivity (σ) of the sample, with 0.2 g of rGO-A obtained by the measurement system. The resistance and pellet thickness values at the given applied pressure were measured using a digital multimeter and a thickness gauge, respectively. The surface resistance (ρs) was calculated by multiplying the resistance (R) by the geometrical correction factor (F) ρs calculation29. The resistivity (ρ) and conductivity (σ) of the pellet are calculated by Eqs  measured by the thickness gauge in the apparatus, and F is a geometrical correction factor.After confirming by microscopic and spectroscopic methods that no structural changes were present in the samples, electrical measurements on the three types of graphene flake samples were conducted to obtain several parameters, such as sheet resistance, resistivity, and conductivity. The schematic illustration and images of the measurement system are depicted in Fig. mentclass2pt{minimThe thickness as a function of the applied pressure was then measured in order to determine the intrinsic electrical behaviour the samples. Since the thickness of a sample pellet at the applied pressure depends on the dose of the sample, thickness measurements were performed on two different pellets made with 0.1 g and 0.2 g of rGO-A at the initial test. The plots of the thickness, resistivity, and conductivity as a function of the applied pressure are shown in Fig. dv) at a given applied pressure is calculated from the mass of the pellet and its geometric dimensions as described in Eq.  or volume conductivity (σv), which is considered taking the geometric dimensions into account, is an intrinsic property of a graphene flakes.Figure d in Eq. 25 as folThis was also found in the analysis of the graphene nanopowder (GNP) sample. Figure Electrical analyses of rGO-B (1.0 g) were performed, and the applied pressure and the pellet density dependent measurement parameters are displayed in Figs 35. To quantitatively and qualitatively analyse the chemical components in the graphene flake samples, the samples were studied by X-ray photoelectron spectroscopy (XPS). The summary of the binding energies and quantitative analysis data obtained from the survey XPS spectra are listed in Table 2 carbon in the graphite component  were purchased from Graphene Supermarket Inc., (USA) and Graphenea Inc. (Spain), respectively. Graphene nanopowder  was also obtained from Graphene Supermarket Inc. (USA). The powder-type graphene materials were used after vacuum drying at 80 °C for 24 hours in order to remove the remaining water content in the sample.A powder resistivity measurement system  was used for the volume resistivity measurements. The measurement system consists of two components: i) a pelletizer and electrode unit and ii) an electrical measurement system. The details of the measurement system are shown in Fig. A dose of the pretreated graphene flake in a vacuum oven, ranging from 0.1 g to 1 g, was transferred into a pelletizer. First, the 4-probe head (ii) and the cylinder (iii) were assembled by inserting the former into the latter and the pretreated sample was charged inside the cylinder. Then, after tapping the sample cylinder in order to obtain a uniform distribution of the powder for a flat surface, the piston (i) was inserted in the cylinder charged with the sample. Finally, the sample-charged pelletizer was connected to the electrode unit, and then pressure varying from 2.6 to 52 MPa was applied to the pelletizer. While pressurizing, the resistance values were recorded at the given pressures using the 4-probe system. Simultaneously, the pellet thickness was measured for the calculation of the pellet’s resistivity. The measurements were performed on three different samples to confirm the reliability and reproducibility of the data sets and between the data sets.α, 1486.6 eV) at 12 kV and 72 W.The morphologies of graphene flake before and after applying the pressure were characterized by ultrahigh-resolution field emission scanning electron microscopy (UHR FE-SEM) using a Hitachi S-5500 instrument and by field emission-transmission electron microscopy (FE-TEM) using a JEOL JEM 2100 F instrument. The structural characteristics were investigated using Raman spectroscopy  that was performed at an excitation wavelength of 532 nm. X-ray photoelectron spectroscopy (XPS) studies were performed using a K-ALPHA+  system with an aluminium anode (Al KSupplementary Information


Thermal radiation can be substantially enhanced in the near-field scenario due to the tunneling of evanescent waves. Monolayer graphene could play a vital role in this process owing to its strong infrared plasmonic response, however, which still lacks an experimental verification due to the technical challenges. Here, we manage to make a direct measurement about plasmon-mediated thermal radiation between two macroscopic graphene sheets using a custom-made setup. Super-Planckian radiation with efficiency 4.5 times larger than the blackbody limit is observed at a 430-nm vacuum gap on insulating silicon hosting substrates. The positive role of graphene plasmons is further confirmed on conductive silicon substrates which have strong infrared loss and thermal emittance. Based on these, a thermophotovoltaic cell made of the graphene–silicon heterostructure is lastly discussed. The current work validates the classic thermodynamical theory in treating graphene and also paves a way to pursue the application of near-field thermal management. Though monolayer graphene has the potential to be used in near-field thermal management applications, no experimental verification has been provided to date. Here, the authors directly measure plasmon-enhanced near-field heat transfer between graphene sheets on intrinsic silicon substrates. For polar dielectrics , this enhancement effect will be more obvious due to the excitation and participation of bounded phononic surface polaritons that will strengthen the electromagnetic coupling across the gaps3. Thermal fluctuating electrodynamic (TFE) theory has been developed to characterize the near-field heat transfer process, initially with analytical formulas describing regular shaped objects  and more recently with sophisticated numerical schemes such as fluctuating surface current formalism6 or quasi normal mode analysis7 that are able to deal with objects with more complex geometries. The continuum theory of fluctuating electromagnetics, as recently predicted by Chiloyan et al.8, will fail to describe the heat transfer over sub-nanometer gaps where direct phonon tunneling will replace thermal radiation to dominate the heat conduction. Various applications based on the framework of near-field heat transfer have been proposed such as thermophotovoltaic (TPV) cell9, thermal scanning imaging10, thermal condenser or rectifier11, etc. On the other hand, the experiment on the near-field thermal radiation has been long delayed due to the technical difficulties primarily in controlling the gap distances although the first measurement was reported in 197012. The super-blackbody radiation was only experimentally observed till 2008 by Hu et al. between two glass plates at a fixed gap distance of 1600 nm13. In recent years, there has been increased attentions and various advanced technologies have been developed to verify the conception of super-Planckian radiation such as between metals16, doped semiconductors18, polar dielectrics16, or metamaterials19, with fixed or variable gaps in the configuration of either microsphere (or point)-plate21 or (micro-) plate–plate16. Quite recently, Cui et al. has pushed the gap limit down to sub-nanometers using the state-of-the-art measurement apparatuses22, while Bernardi et al. has successfully characterized the near-field heat flux between two macroscopic silicon wafers at nanoscale gaps23.Objects with temperature will radiate the infrared light arising from the mechanical oscillation of inner charges and the emitted light spectrum can be statistically characterized by the classic Plank’s radiation law. For the far-field scenario, a blackbody that absorbs all the impinging light has the capacity to yield the maximum energy transfer efficiency compared with nature materials. When a receiver is placed at a distance far smaller than the thermal de Broglie wavelength  as the single-carbon atomic layer material has unique plasmon polariton response in the infrared window which provides a promising material option to manipulate the excitation and emission of thermal photons. Compared with highly doped plasmonic semiconductors, graphene has the outstanding advantages including tunable Fermi level with a linear Dirac band, for example, by voltage-gating, easy transfer to or be integrated with different substrates and good thermal stability. Theoretically, enhancement of the heat transfer efficiency by graphene33. Thermal radiation with power density far larger than the Blackbody limit is first observed with intrinsic Si substrates (negligible loss) as assisted by the inter-layer EM coupling. In a comparison, the heat transfer between the bare Si–Si substrates is fractionally small. The measurement is repeated using highly doped silicon substrates that alone can give rise to strong heat transfer in a near-field pair as material loss and evanescent wave tunneling increase. Covering their surfaces with Gr sheets could further enhance the heat transfer efficiency although in this case, graphene will exhibit a lower Fermi level resulted from the electron doping over the junction. The measured results are well reproduced analytically with the input of the measured material properties, thus explicitly validating the classic TFE theory. The current work also provides a useful material structure and measurement platform to explore the full features of Gr plasmons controlled thermal photon manipulations, which is specifically discussed in the end for TPVs25.In this work, we managed to have a direct measurement of plasmon-mediated near-field heat transfer between two graphene sheets with nanoscale vacuum gaps. Silicon substrates with well-controlled physical properties has been utilized to host the graphene layer. The heterostructure also provides a platform to modify the charge density of graphene by forming a Schottky junction at the Gr–Si interface34. The sample is cut into 2×2 cm squares for experiment. All these processes including the measurement are conducted in a cleaning environment. The details are addressed in the Methods section. Silicon hosting substrates are employed here primarily for three reasons. First, their controllable infrared properties through doping can be tailored to identify the Gr plasmon’s role in the near-field heat transfer. Second, silicon wafer with an ultra-small bow is achievable 35. And lastly, the Gr–Si heterostructure may be a potential assembly for the important TPV or photothermoelectric applications. They have a roughness about 1–2 nm and a maximum bow value less than 20 nm determined by a Laser interferometer (ZYGO OMP-035/M) with an area of 2×2 cm. Figure −6 Torr) assisted by four AZ photoresist (PR) posts . The PR posts have good physical stability below 150 °C and their height decides the gap value. In our setup, the top Gr/Si assembly (temperature Te) behaves as the emitter sourced by a same sized heating plate through a copper spreader and the bottom Gr/Si assembly (temperature Tr) is the receiver sunk by a temperature electric controller (TEC)  through another copper spreader. Silicone grease is used to improve the boundary thermal contact between the stacks. A 200-nm thick titanium film has been deposited on the top surface of the silicon substrate in order to block the photons emitted from the grease layer. Two temperature sensors are inserted into the center of the top and bottom copper spreaders through the drilled slots to monitor Te and Tr. A pair of magnets consisting of a 5×5×3 cm3 NdFeB hard magnet  and a 2×2×1 cm3 iron soft magnet  are used to strengthen the contact and mechanical stability of the system by forming a clamping force of about 10 N. The exposed surface of the top half unit is coated by a 200-nm thick aluminum film to minimize the background radiation. In the measurement, we have assumed the Gr/Si assembly has the same temperature with the copper spreader since the contacting thermal resistance is negligibly small compared with the radiation one. The Gr/Si/Cu triplets at the receiver side are thermally maintained at a stable temperature around 30 ± 0.30 °C  assisted by the TEC cell. The maximum temperature difference (ΔT = Te − Tr) we obtain is ~50 °C at a vacuum gap around 500 nm and ~70 °C as the gap is larger than 1 µm.The monolayer graphene used in the work is a commercial product made by chemical vapor phase deposition (CVD)  originally grown on a copper foil. Standard technique has been employed to transfer the macroscopic Gr sheets onto silicon substrates which are adhered together by the Van der Waals forcePin after subtracted by the resistive heating loss of electrode wires is majorly dissipated through two channels: one we called is the background power loss (Pb) including one part conducted along the electrode wires of source and temperature sensors and the other part radiated toward the surrounding space and the second is the net power (Pr) radiated via the vacuum gap. As the total input power is known, it is crucial to correctly evaluate the background transfer branch Pb, which in our case is done in a separate experiment measuring the temperature rising in a far-field configuration. As schematically shown in Fig. Pb at different Te is estimated through a simple relation: Pb(Te) = Pin(Te) − PrFar(Te), where PrFar = εσ(Te4 − Tb4) (ε-emissivity and σ-coefficient) is the power emitted via the Gr/Si surface. As Te is relatively small, the radiation via other Al-film coated surfaces is neglected, i.e., assuming that Pb is primarily caused by the heat flowing out along the electrode wires. Figure Pin(ΔT) = a × ΔT + b × ΔT4 with the fitting coefficients a = 0.014 W K−1 and b = 5.2 × 10−9 W K−4. Note here we use ΔT = Te − Te0 and Te0 = Tb = 30 °C. When ΔT < 40 °C, the temperature is linearly dependent on the input power, indicating that the background radiation could be neglected at small temperature changes. Thereafter, in the following, the near-field radiated heat is evaluated by the equation Pr = Pin(ΔT)−a × ΔT − PAZ(ΔT), where PAZ is the heat conducted via the four posts.As sketched in Fig. 2. Statistical measurement shows the surface roughness of our sample is less than ~50 nm, which is relatively small when considering the multiple physical and chemical processes involved in the sample preparation. As a consequence, in the experiment, we set the height of the photoresist posts or gap size at values larger than 400 nm. Figure −1, respectively. Their small peak intensity ratio indicates the high quality of the CVD graphene36. According to the G-mode position, we can estimate the Fermi level of graphene at EF = 0.27 eV due to the electron doping from the absorbed molecules, which is close to the previous reported value (0.32 eV) also for a CVD graphene37. In addition, the strong single 2D peak identifies the dominant monolayer nature of our graphene sample38. Figure TGr and Ti-Si denote the transmittance with and without graphene, respectively. There is a broad peak at 4832.8 cm−1, associated with doping inhomogeneity, temperature effect and other broadening mechanisms. The spectrum has no obvious change as we raise the temperature from 30 to 70 °C, indicating the thermal stability of the heterostructure. The middle-IR broad absorption curve can be phenomenologically fitted by a Gaussian step function37. From the full width at half-maximum (FWHM), we can estimate the “2EF onset” of the inter-band transition, as indicated by the arrow in Fig. d of the silicon substrates is mainly decided by the height of the photoresist posts. The real value may have a certain distribution due to the bow of wafer or roughness, etc. As shown in Fig. d represent the edges of the experimental lines measured at various spatial points. Figure Our first experimental sample utilizes an insulating silicon (i-Si) substrate  without doping so that the substrate contribution to the near-field heat flux could be minimized and the role of the Gr plasmons can be more clearly examined. The maximum temperature change for the emitter is less than 100 °C. The variation of infrared properties due to temperature change for both silicon and graphene can be practically neglected. Figure h varied as a function of temperature difference ΔT for the samples with and without graphene. The measured gap size is 430 ± 25 nm and the receiver’s temperature is controlled at Tr = 30 °C. For the bare Si–Si assembly, the measured heat flux density (blue dot) fluctuates below the line (black) representing the Blackbody radiation. Although the intrinsic silicon has an ultra-small imaginary permittivity (~10−4 derived from the FTIR absorption measurement), the thick substrate will still produce certain amount of thermal radiation and this effect will be enlarged in the near-field case with the involvement of evanescent wave tunneling23. The heat flux from the naturally oxidized ultrathin SiO2 layer  on the silicon surface is negligibly small. The blue dashed line in Fig. T = 36 °C, the heterostructure gives rise to a strong flux density at h = 1195 W m−2, nearly 4.5 times larger than the Blackbody radiation (h = 268 W m−2).The total radiation heat flow at this case is about 0.48 W for our sample, which is nearly four times larger than that (PAZ ~ 0.12 W) conducted along the four supporting posts. The red shaded region profiles the theoretical heat flux distribution calculated by considering the fluctuations: d = 430 ± 25 nm and EF = 0.27 ± 0.03 eV. The collision time factor for a CVD graphene used here is τGr = 100 fs40. The experiment data is generally well reproduced by the theory. Here we may overlook the radiation contribution from the surface residuals but they do have indirect influences by affecting the Fermi level of graphene and their near-field coupling strength.Figure p-polarization photon transmission probability τp  across the vacuum gap without and with graphene at ΔT = 50 °C and d = 500 nm, respectively41. The metallic ground is described by a Drude model with the data input from ref. 42. Usually, the p-polarized contribution is dominant in the near-field flux43. Without graphene, only traveling waves inside the free-space light cone (from y-axis to the line ω = ck) has a high transmissivity, in particular for those near the edge of the light cone that have longer light paths in the substrate. The inhomogeneous transmission pattern reflects the Fabry–Perot (FP) resonance features of our low loss planar system. The tunneling possibility for evanescent waves inside the silicon light cone (defined by the line ω = v/k with v the light velocity in silicon) is rather small, which will become obvious only using much thicker substrates or at smaller gaps (<100 nm). When a graphene layer is introduced, as shown in Fig. 44. In addition, there appears a new and p-polarized evanescent wave band below the silicon light cone arising from the near-field EM coupling in an asymmetric super-mode pattern  n-type silicon substrates that can strongly modify the dielectric properties of the background and grapheme layers. Especially, the doped silicon (d-Si)/Gr heterostructure is interesting for the potential applications in TPV cells or thermophotoelectric sensors due to the formation of a Schottky junction at their interface46, which will modulate the infrared response of graphene as we observed before31. The dielectric properties of the heterostructure are derived from the measured FTIR spectrum. Further information is given in the Methods section. Figure 47. Then, a Drude model fitting  accompanied with a dramatic change on both real and imaginary permittivity. Figure 4b48. From terahertz to far-IR, the monolayer graphene will also electromagnetically behave as an optical metal and can be described by a Drude model conductivity resulted from the intra-band electron scattering. In Fig. EF = 0.15 ± 0.03 eV. The EF value on the d-Si substrate is nearly half of that (0.27 eV) of the intrinsic silicon, indicating the occurrence of electron transfer from the d-Si substrate to the p-type graphene  assisted by the Schottky heterojunction at the Gr–Si interface49.In the second experiment, we will repeat the experiment using doped . The increment becomes obvious when the gap is shrunk to 430 nm. At an example point of ΔT = 37 °C, the heat flux density increases about 11% after introducing graphene. The measured results are consistent with the analytical predictions (shaded regions) calculated using the measured material and structural parameters. In addition, it is noted that with the same configurations, the d-Si/Gr heterostructure pair will give a much larger heat flux than the i-Si/Gr pair, primarily due to the loss-induced additional channel for the generation and transmission of thermal photons using doped silicon.Figure p-polarization transmission probability τp  between the d-Si pairs without and with graphene at ΔT = 50 °C and d = 500 nm, respectively. For the bare d-Si pair, compared with that shown in Fig. ω = v/k, arising from the plasmonic near-field coupling between the silicon (negative ε in this region) surfaces. A typical asymmetric field pattern for this super-mode is shown in Fig. d > 100 nm)45. On the other hand, the momentum and mode pattern matches will improve the near-field coupling of plasmonic polaritons between graphene and silicon substrate. As a consequence, more evanescent waves can be transferred to strengthen the heat flux, as shown in Fig. Figure 51 and heat transfer efficiency has been experimentally observed at different substrates. The results not only validate the continuum theory of fluctuating electromagnetics in dealing with the single layer material, but also provide an indirect evidence about the existence of this highly bounded surface modes and their tunability in our macroscopic characterization. For this aim, it is crucial to have high measurement accuracy and good quality sample. The former is achieved by our custom-made setup as evidenced by the high agreement of the measured and calculated heat flux densities between the bare d-Si substrates  with an interface p–n junction in order to excite and separate electron–hole charges52. From the material perspective, the Gr–Si heterostructure with the existence of Schottky junction could be more realistic compared with the p–n junction which for far-IR optics is not readily available yet. The tunability of Fermi level of graphene will also help to modulate the barrier height and improve the utilization of thermal photons, which becomes critical when the working temperature is relatively low. Direct integration with semiconductors is also advantageous for other device applications such as thermal rectification53 or thermoelectric sensor55. In addition, high level chemical doping using species like HNO3 may be employed to further tune the Fermi level of graphene with larger modulation depth56 and the number of graphene layers may be optimized to strengthen the near-field coupling and further enhance the heat flux. These methods need more and deep investigations in future.It needs to be pointed out that silicon may not be the best choice to host grapheme if one wants to maximize the heat flux and for this aim polar materials such as silica or BN may be used where the occurrence of plasmon–phonon hybridization can further strengthen the tunneling of evanescent wavesT1 and T2 (=30 °C), respectively. The TPV cell is specifically biased to have a potential difference Vb in order to maximize the output power. A typical interface band diagram for the graphene-covered n-type silicon is drawn in Fig. WGr is the work function of graphene and χ is the electron affinity in silicon. Ec (Ev) and T1 = 700 °C and d = 50 nm. The discontinuity of the curve for the cell side is caused by the excitation of non-thermal photons arising from the potential difference57, with more details addressed in the Methods section. Under the current device parameters, the main part of absorbed thermal photons are attenuated into heat. Smaller gaps or higher temperature will help to increase the efficiency of photon utilization but at the cost of practical challenge25. In Fig. Prad received by the cell and the portion PGr absorbed solely by the graphene layer on both insulating and doped silicon substrates as ΔT ranges from 100 to 700 °C. At this small gap (50 nm), lower loss for the substrate will be more favorable to strengthen the heat flux as the coupling of the graphene layers will be stronger. The thermal photons from the emitter are dominantly absorbed by the graphene layer, which is nearly affected by the loss degree of the substrate. In Fig. PPV and efficiency η ( = PPV/Prad) of two Si/Gr TPV cells at different silicon doping. As a comparison, we also give the result of an InSb (band gap = 0.17 eV57) based semiconductor p–n junction at the same gap (50 nm). For both cases, we assume an ideal photocurrent responsivity of 100%  will further enhance the evanescent wave tunneling and lead to a larger heat flux density. The unique property of the Si/Gr heterostructure may provide a promising platform to explore the applications of near-field heat transfer systems for various purposes, in particular for TPVs.58 to prepare the i-Si/Gr and d-Si/Gr heterostructures in the following steps. The graphene sheet is first peeled off from the polymer holding substrate and floats above the DI water for more than 2 h before transfer. Silicon substrates are handed using a tweezer to pick up the floating graphene. Great carefulness should be paid to avoiding air bubbles or chemical contamination for Gr sheets. After this, the sample is dried naturally for more than 30 min and then baked for 20 min at 100 °C. Finally, we rinse the sample in a 45 °C acetone for 30 min and then dry it in a 50 °C oven for 10 min. On the surface of the Si/Gr heterostructure, the photoresist  is spin coated at 4000 rpm for 29 s and then baked for 6 min at 90 °C. It will lead to an about 1150 nm photoresist layer. A standard UV lithography procedure is employed to fabricate the four supporting posts. The ion coupled plasma (ICP) etching has been used to thin and control the post height for samples with small gaps.The double-side polished Si wafers are used and cut into 2×2 cm pieces of substrates before transfer graphene. They are cleaned by ultrasonic waves sequentially in acetone, methyl alcohol, and isopropyl alcohol. Each step is for 5 min. After these, a 200 nm titanium film is evaporated onto the backside surface of the Si substrates, which are followed by a 500 °C post-annealing for 5 h to release the stress that helps to reduce the bow of substrates.We utilize the mature technique called Trivial Transfer Graphene (TTG)23. Using the setup described in Fig. For the temperature measurement, the Platinum Resistance Temperature Detector  is used to monitor the temperature of the emitter and the receiver. We use a source meter  to measure the resistance of thermal sensor. The heater is powered by a DC power source . Its voltage is slowly increased from 0.5 to 3.0 V at a time interval of 15 min between two successive steps, corresponding the total input power changing from 0.05 to 1.05 W. In the process, the receiver is maintained at a constant temperature of 30 °C with the help of TEC. For our firmly clamped near-field system and the usage of conductive grease layer at the boundaries, it is reasonably assumed that the measured copper temperatures are equal to the Si/Gr emitter and receiver59. For the Si pair without graphene, the near-field radiative heat flux is calculated by:τs,p is the transmission probability of the p-polarizations and s-polarizations, derived asi = 0 represents the vacuum gap and j = 1 or 2 denotes the emitter or receiver.When a monolayer graphene is introduced on silicon, the Fresnel reflection coefficient in Eq.  denotes the normal component of wave vector in vacuum or dielectric, μ0 stands the permeability of vacuum.The near-field thermal radiation is calculated using the classic fluctuating electrodynamics theoryentclass1pt{minima14ωp,h and γh (γe) represent the plasma and collision frequencies for free carriers of hole (electron), respectively. We measure the reflectance of the doped silicon by FTIR at a 13° incident angle. Such a small incident angle will give a small coefficient change compared with those at the normal incidence. The measured reflectance spectra at different sample temperature are plotted in Fig. 8 rad s−1,13 rad s−1, 14 rad s−1, and 14 rad s−1. Based on these input, the permittivity spectra of the doped silicon are plotted in Fig. EF = 0.15 ± 0.03 eV.The doped silicon has a tunable plasma frequency dependent on the carrier concentration. Typically, it can be described by the Drude model given belowVb (=Φb/e) will impose a chemical potential μc on the emitted photons with energy larger than the barrier and in this case, the PV cell will give a backward radiation power to the emitter by57:μc = Φb for ω > ω0 and 0 for ω < ω0. The emitter’s radiation still follows Eq.  Φb/e. In our calculation of Fig. PPV = Iph × Vb. The power efficiency of the cell is defined by PPV/Prad. In our calculation, the parameters for i-Si are same with those used in Fig. −1 and −1. In the two cases, EF = 0.5 eV and τGr = 100 fs for Gr. The parameters for InSb are obtained from ref. 62.For the PV cell, the potential difference mentclass2pt{minimPeer Review File


Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-β pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both  IPF patients exhibit symptoms such as a persistent cough and dyspnea, and lung function deterioration continues in these patients to the point of respiratory failure1. The pathogenesis of IPF is still unclear but in recent years consensus has focused toward a hypothesis that this disease is the result of repeated injury and/or genetic predisposition to dysfunction in alveolar type-2 epithelial cells (AEC)2. Sources of AEC injury include smoking, particulate inhalation, viral infections, and gastroesophageal reflux2. In response of AEC injury, fibroblasts are recruited to the site of injury and the recruited cells are subsequently activated via TGB-β to become myofibroblasts, which secrete excessive amounts of extracellular matrix (ECM) proteins3. Current approved therapeutics for IPF include nintedanib and pirfenidone (i.e. standard-of-care (SOC) drugs), and these therapeutics appear to have direct and indirect effects on myofibroblast activation4. However, adverse side effects and the failure of these drugs to halt progression in IPF highlights the need for a more effective treatment(s)6.IPF is a devastating disease, characterized by the unrelenting spread of lung fibrosis in a honeycomb pattern throughout the lung parenchyma and a median survival of 3–5 years after diagnosis8. Mechanistically, MAP3K19 appears to modulate TGF-beta signaling via the translocation (or shuttling) of activated Smad2 and Smad3 into the cell nucleus8. In a mouse model of smoke-induced emphysema, both an anti-MAP3K19 siRNA and a small-molecule inhibitor directed at this MAPK inhibited inflammation and improved airspace integrity7. Targeting MAP3K19 also strongly inhibited bleomycin-induced pulmonary fibrosis when administered either prophylactically or therapeutically8. In the current study, we report the effects of both siRNA and novel small-molecule inhibitors of MAP3K19 in primary human IPF fibroblasts and a SCID mouse model of IPF induced by the intravenous injection of IPF fibroblasts10.MAP3K19  is a conserved stress-induced protein, which is upregulated in alveolar and interstitial macrophages, bronchial epithelial cells, and type II pneumocytes in COPD and IPF11. Patient characteristics have been previously published in detail12.The current study was reviewed and approved by an Institutional Review Board at the University of Michigan Medical School . Informed consent was obtained from all patients who participated in the study described herein. Methods pertaining to patient samples were all conducted in accordance with all relevant guidelines and regulations. Patients were evaluated before undergoing fiberoptic bronchoscopy and clinical evaluations included chest radiography, lung function measurements, and thin section computed tomography12. Primary normal lung fibroblasts were derived from non-fibrotic lung samples lacking any evidence of disease. Histologically-proven IPF and normal lung samples were dissociated mechanically as previously described in detail13. Briefly, SLBs were mechanically dispersed in 150 cm3 flasks (Corning), combined with Dulbecco’s modified Eagle Medium (Lonza), containing 15% fetal bovine serum (Cell Generation), 0.1% Pen/Strep/Amphotericin B  (Lonza), 0.1% (29.20 mg/mL) L-glutamine (Corning), and Primocin at 100 μg/ml (Invivogen). Isolated fibroblasts were incubated at 37 °C in 10% CO2 until confluency was reached. The cells were passaged 3 to 4 times before use in the experiments described herein. In the present study, we analyzed a total of 4 normal fibroblast lines, 7 fibroblast lines derived from IPF patients who showed slow progression, and 7 fibroblast lines derived from IPF patients who showed rapid progression.Lung fibroblasts were derived from surgical lung biopsies (SLBs) from patients who exhibited slowly or rapidly progressing IPF  over the first year after diagnosisIn vitro IC50 of this first-generation inhibitor directed against MAP3K19 was determined to be 177 nM. Testing this MAP3K19 inhibitor in a panel of 358 kinases, AXP1741 was found to partially inhibit PDGFRA and B, and YES1 at 10 μM. In PK studies with this compound the following was observed: Cmax 42 ng/ml; T1/2 32 hour; AUC 1163 μg*hr/L. Finally, AXP1741 was found to be efficacious in an A549 cancer xenograft model at 10 mg/kg in mouse (unpublished findings).Molecular mass is 302.34 g/mol. in vitro IC50 of this second-generation compound directed against MAP3K19 was determined to be low, at 435 nM, and the compound possessed sub-optimal PK properties so was not characterized further.Molecular mass is 436.52 g/mol. The In vitro IC50 of this third-generation inhibitor directed against MAP3K19 was determined to be 8.7 nM ). In PK studies with this compound the following was observed: Cmax 351 ng/ml; T1/2 2 hour; AUC 2050 μg*hr/L Finally, AXP2258 was also found to be highly efficacious in an A549 cancer xenograft model at 10 mg/kg and its effect was additive with Nexavar (unpublished findings).Molecular mass 408.46 g/mol. 14. Cultures of an IPF fibroblast line with the highest transcript expression of MAP3K19 (line S117) were grown in 150 cm2 tissue culture flasks, and trypsinized to generated single cell suspensions. These isolated cells were then labeled with PKH26, as previously described9, and the labeled fibroblasts were transferred to a phosphate-buffered saline (PBS) solution at a concentration of 2 × 106 cells/ml. Five hundred (500) μl of suspended fibroblasts was injected into the tail vein of each C.B-17SCID/bg mice. Groups of five C.B-17SCID/bg/group received one of the following at indicated times and doses following human fibroblast injection: (1) siRNA directed against human MAP3K19 ); both administered at 5 mg/kg intranasally every other day starting at either day 14 or 30 after human IPF fibroblast injection; (2) non-sense siRNA ) both administered at 5 mg/kg intranasally every other day starting at either day 14 or 30 after human IPF fibroblast injection; (3) PBS  every other day starting at either day 14 or 30 after human IPF fibroblast injection; (4) interleukin-13/Pseudomonas exotoxin chimeric protein  every other day starting at either day 14 or 30 after human IPF fibroblast injection; (5) vehicle used to suspend compounds (40% solutol + 10% ethanol + 50% H2O) orally; (6) AXP1741 (10 mg/kg) daily orally; (7) AXP2132 (10 mg/kg) daily orally; or (8) a multi-JAK inhibitor Tofacitinib (1 mg/kg) daily orally (as control for the off-target activity of AXP2258 directed at JAK1 and JAK3); and (9) AXP2258 (1 mg/kg) daily orally. All mice were euthanized by anesthesia overdose at days 60 or 63 after injection of human IPF fibroblasts, and whole lung tissue was dissected and used for subsequent analysis of histologic, molecular, and protein parameters.Female, C.B-17-scid-beige  were purchased from Taconic Biosciences , and maintained in barrier facility at the University of Michigan Medical School. All methods were performed in accordance with guidelines and regulations for animal care and use at the University of Michigan School of Medicine. All mice had access to autoclaved water and pelleted chow diet. All procedures with C.B-17SCID/bg mice were performed in a sterile environment and these procedures were approved by an animal care and use committee at the University of Michigan Medical School. This adoptive transfer model of lung fibrosis is initiated by the intravenous introduction of cultured IPF fibroblastsFollowing euthanasia, right lobes of each C.B-17SCID/bg mouse were dissected and infused with 10% formalin solution, then placed into a fresh formalin solution for 24 hours. Lungs were paraffin-embedded using a standard technique. Lungs were sectioned at 5 μm and stained for hematoxylin and eosin, and Masson’s trichrome for architecture analysis.RNA was isolated from cultured normal and IPF (both from slow and rapid progressing patients) fibroblasts as well as whole-lung samples using RNeasy kits (Qiagen). Isolated RNA was reverse transcribed into cDNA using SYBR Green PCR Master Mix (Applied Biosystems) and 5 ng of cDNA was used for transcript quantification via RT-PCR using a ViiA 7 (Applied Biosystems). Oligonucleotides primers were designed and validated by Integral DNA Technologies. MAP3K19 primers were obtained from Axikin: sense primer: 5′AATGGCACCCACAGTGACATGCTT3′; antisense primer: 5′CCCTCGGTGTGCTCCGATCTAAAA3′. The amplification efficiencies were determined for target genes and normalized to GAPDH. Delta Ct was used for comparative analysis. Commercially available primer and probe sets for both human collagen 3 and fibronectin was obtained from Applied Biosciences (Thermo Fisher). Fold change and gene expression were analyzed with Data Assist software (Life Technologies).17. β-tubulin values were used to normalize values of αSMA and Collagen 1.Alpha smooth muscle actin (αSMA) and β-tubulin proteins were measured using an in-cell ELISA technique. Cell-free supernatants (50 μl) from cultured fibroblasts were used to measure collagen type 1 levels, using a direct ELISA, as previously described10. Briefly, whole-lung tissue was homogenized . Five hundred (500) μl of homogenate samples was hydrolyzed in 1 ml of 6 N HCl, for 8 h at 120 °C. For each sample, 5 μl of sample combined with 5 μl of citrate/acetate buffer  and 100 μl of chloramine-T solution . Solution samples then were incubated at room temperature for 20 min, after which 100 μl of Ehrlich’s solution (Aldrich) was added. After incubating in Ehrlich’s solution for 15 min at 65 °C, the samples were analyzed with a Beckman DU 640 spectrophotometer at a wavelength of 550 nm. Sample hydroxyproline concentration was calculated from a standard curve (0–100 μg/ml).Total lung collagen concentration was calculated using a technique previously describedAll statistical analyses were conducted using GraphPad Prism 7. Data are presented as mean ± SEM. Statistical significance, when comparing two groups, was determined using nonparametric t-tests (either Mann-Whitney or Kolmogorov-Smirnoff) to compare differences between IPF (Stable/Rapid) groups and normal lung groups. When comparing more than two groups, Ordinary One-way ANOVA tests were used. P values considered significant; *p < 0.05, **p < 0.01, ***p < 0.001.MAP3K19 transcript levels in diagnostic biopsies from IPF patients were significantly elevated when compared to normal lung samples, Fig. . IPF fibin vivo, IPF fibroblasts were injected into C.B-17SCID/bg mice as previously described in detail12. Beginning at either days 14 (during the development of fibrosis9) or 30 (when fibrosis is established9) after IPF fibroblast injection (line S117A), C.B-17SCID/bg mice received PBS, IL13-PE, scrambled siRNA (n/s), or siRNA targeting either mouse or human MAP3K19 by intranasal delivery every other day until day 60 after human fibroblast injection. As shown in Fig. To address the role of MAP3K19 role in fibrosis In the next series of experiments, two small-molecule inhibitors of MAP3K19, namely AXP1741 and AXP2132, were tested in a humanized mouse model of IPF. In these studies, stable IPF fibroblasts (line S117A) were injected into C.B-17SCID/bg mice and beginning at day 35 after fibroblast injection, groups of 5 mice received one of the following every other day until day 63 after fibroblast injection: PBS , IL13-PE , compound vehicle , AXP1741  and AXP2132 . Although AXP1741 therapy significantly reduced hydroxyproline compared with the oral vehicle control Fig. , it was We undertook additional studies with a more potent inhibitor called AXP2258, and also explored the combinatorial effects of AXP2258 and either BIBF1120 (i.e. nintedanib) or pirfenidone on human lung fibroblast activation via the measure of both αSMA and collagen 1 protein. AXP2258 had no effect on generation of either αSMA and collagen 1 protein in normal lung fibroblasts compared with the DMSO vehicle control Fig. . HoweverThe combination of AXP2258 and the SOC compound nintedanib had no effect on either myofibroblast protein marker compared with the nintedanib alone group in cultured normal lung fibroblasts Fig. . When AXThe last set of experiments addressed the therapeutic effects of a third inhibitor of MAP3K19, namely AXP2258, which was administered from days 35 to 63 in a C.B-17SCID/bg model of IPF. Compared with the appropriate controls and two other treatments (IL13-PE (1 μg/dose) and Tofacitinib ), AXP2258 (at 1 mg/kg) significantly reduced the total lung concentration of hydroxyproline at day 63 after fibroblast injection Fig.  compared2. Nintedanib and pirfenidone are two FDA-approved drugs that slow but do not stop progression in this disease19. This benefit is not without serious tolerance issues as evidenced by the debilitating side-effects IPF patients experience while taking either or both of these therapeutics20, thus providing the impetus to pursue other therapeutic strategies in IPF. Previous studies had indicated that targeting MAP3K19 in the context of experimental smoke-induced7 and bleomycin-induced8 lung injury and remodeling was therapeutic in both models via mechanisms involving inflammation and TGF-β-induced remodeling. Consequently, the aim of our study was to further explore the role of MAP3K19 in IPF and determine its role in primary human lung fibroblasts and in a C.B-17SCID/bg model of IPF. We show that MAP3K19 was highly expressed in IPF fibroblasts particularly those derived from IPF patients who exhibited stable or a slowly progressive IPF in the first year after diagnosis (as previously defined12). Targeting MAP3K19 both in vitro and in vivo markedly altered IPF fibroblast to myofibroblast differentiation and the maintenance of pulmonary fibrosis in C.B-17SCID/bg humanized with IPF fibroblasts. Thus, these data demonstrate that MAP3K19 modulates fibrosis via direct effects on primary human IPF fibroblasts.It is hypothesized that repetitive and unrelenting injury to the lung is a major mechanism driving the fibrotic process in IPFet al.12 and it incorporates both changes in both forced vital capacity (FVC) and diffusing co-efficient of carbon monoxide (DLCO) as discriminators in these IPF patient subgroups. Our in vitro analysis of slow IPF fibroblasts confirmed that MAP3K19 was upregulated in these cells. At present, it is not clear as to why the expression of MAP3K19 was higher and regulated in slow IPF fibroblasts and not in rapid IPF fibroblasts. Our previous studies have documented considerable differences between lung fibroblasts cultured from these subsets of IPF patients including differences in responses to hypomethylated DNA21, double-stranded RNA22, and interleukin-4 & 13 (IL-4/IL-13)14. For example, we have observed that slow IPF fibroblasts are less responsive to IL-4/IL-13 compared with rapid IPF fibroblasts23 and these findings were confirmed in the present study with the observation that the immunotoxin IL13-PE had no anti-fibrotic effects in the humanized SCID model of IPF induced by the introduction of stable IPF fibroblasts. In previous studies, we have observed that IL13-PE was selective and only targeted IPF cells with upregulated IL-13Rα224, which is typically observed in rapid IPF fibroblasts. What dictates responsiveness and non-responsiveness to external signals as well as the differential nature of signaling pathways in rapid versus slow IPF remains an active area of investigation. Clearly, further work is required to better define the fibroblast-centric mechanisms that distinguish rapid from slow progression in IPF.Overall analysis of diagnostic biopsies from IPF patients indicated that there was an approximate 3-fold difference in MAP3K19 transcript expression between normal and IPF lung samples. Further exploration of the samples and cells from these samples showed that this increase was restricted to IPF patients who exhibited slow progression or more stable disease in the first year after diagnosis. The criteria we used to define slow and rapid decline in IPF are found in our previous study by Trujillo 25. Specifically, when added alone to cultures of primary lung fibroblasts, AXP2258  significantly inhibited the proteins levels of both myofibroblast markers in cultures of slow IPF fibroblasts compared with the DMSO control group. AXP2258 treatment alone either had no effect or increased α-SMA in cultures of normal and rapid IPF fibroblasts, respectively. Interestingly, the addition of pirfenidone with AXP2258 to cultures of slow and rapid IPF fibroblasts significantly inhibited α-SMA protein expression in these cells compared with pirfenidone treatment alone. Also, the addition of pirfenidone with AXP2258 to cultures of all three human fibroblast types examined in this study significantly inhibited collagen-1 protein expression in these cells compared with pirfenidone treatment alone in our in vitro assays. Unlike with AXP2258 combination with pirfenidone, the combination of AXP2258 with nintedanib only significantly decreased α-SMA protein expression compared with nintedanib treatment alone in cultures of slow IPF fibroblasts. Why there were differences between the effects of combined AXP2258 and the SOCs is not clear at the moment but differences in responses to drug + SOC combinations in IPF trials are beginning to appear in the literature26. Thus, AXP2258 treatment alone inhibited fibroblast to myofibroblast differentiation in fibroblasts from slow IPF patients but when this MAP3K19 inhibitor was combined with pirfenidone inhibitory effects on myofibroblast differentiation were observed in both slow and rapid IPF fibroblasts.Our data suggest that MAP3K19 affects the synthetic properties of IPF fibroblasts to a greater extent compared with primary normal lung fibroblasts, particularly with respect to myofibroblast differentiation, which we defined in cultured fibroblasts as changes in both α-SMA and collagen 1 protein expression8. In the present study, AXP1741 and another second-generation small-molecule inhibitor of MAP3K19, namely AXP2132, therapeutically modulated fibrosis in a humanized C.B-17SCID/bg mouse model initiated by the intravenous injection of stable IPF fibroblasts. More importantly, we noted that the clinical candidate AXP2258 exhibited the strongest inhibitory effect on the fibrosis parameters measured in this model. Although this compound has been shown to also target JAKs, treatment with the JAK inhibitor Tofacitinib had no effect on the pulmonary fibrosis observed in this humanized SCID model of IPF. The in vivo studies in humanized C.B-17SCID/bg mouse model were conducted using a stable IPF fibroblast line because fibroblast lines from this subtype of IPF showed the most consistent expression of MAP3K19. Interestingly, the anti-fibrotic effects of these two small molecule inhibitors in this model were similar to the in vivo effects of siRNA directed at human but not mouse MAP3K19. Although the mechanism via which MAP3K19 modulates fibrosis in the humanized SCID model is not presently clear, we speculate that MAP3K19 works via a TGF-β-dependent pathway, as previously described in the bleomycin model of murine lung fibrosis8. Thus, the exact mechanism via which MAP3K19 regulates fibroblast to myofibroblast transition in IPF patients requires further investigation particularly whether the targeting of MAP3K19 alters lung responsiveness to profibrotic mediators including several growth factors.In a previous study employing a bleomycin-induced fibrosis model, it was shown that therapeutically dosing with AXP1741 at 10 mg/kg significantly reduced the fibrotic response to this injuryin vivo reproducibility, and most importantly adverse side-effects27. MAP3K19 is unique in that it is highly specific to the lung and it is uniquely upregulated in chronic lung diseases with abnormal remodeling8.In conclusion, our studies demonstrate that MAP3K19 has a pro-fibrotic role in IPF. A review of studies addressing the targeting of the TGF-β/SMAD2/3 pathway in fibrosis highlights a consensus that the biggest problem with this approach relates to cell-specificity, Supplementary Figure 1


The crystallization processes of the 3D BFO/graphene composites with different graphene oxide (GO) concentrations were studied for their visible light photocatalytic properties. Compared to the single BFO submicron cubes, 3D BFO/graphene composites have greatly improved photocatalytic activity. A high photocatalytic performance is obtained at a GO concentration of 3 mg/mL, with the degradation rate of methylene blue (MB) dye reaching up to 92% in 140 min. The enhancement of photocatalytic activity can be attributed to the large specific surface area and 3D architecture of 3D composites, which provide more transport paths to effectively improve the separation rate of photo-generated electrons and holes. Therefore, 3D BFO/graphene composites have a broad prospect of application in the field of photocatalysis.Bismuth ferrite (BiFeO For band gap . Effortsband gap , as wellband gap , nanowirband gap ,34, micrband gap , and porband gap . In addimaterial . 3PO4 heterostructures to degrade different dyes with high catalytic activities in the visible light range. Zheng et al. [3N4–TiO2–GA) composites by combining the hydrothermal method with freeze-drying. The adsorptivity for RhB was as high as 96.5% in the dark, and the overall removal efficiency reached up to 98.4% under visible light irradiation within 60 min.Carbon materials have many excellent physical and chemical properties, and are widely used in many fields ,40,41,42g et al.  synthesiNevertheless, little work has been done to systematically study the effects of graphene addition on the morphologies and photocatalytic properties of 3D BFO/graphene composites, and the corresponding mechanism is not clear , which m3)3•5H2O and Fe(NO3)3•9H2O crystals were weighed in a molar ratio of 1:1, and then dissolved in 50 mL of deionized water by magnetic stirring. NaOH solution (2 M) was slowly added into the mixed solution containing Bi3+ and Fe3+ under magnetic stirring, producing a large amount of orange precipitate. Then, 2 mL of hydrogen peroxide (30%) was added into the reactor, and the solution was placed in an environment of 200 °C for 72 h. After the hydrothermal reaction, the reactants were centrifuged 4–5 times at a speed of 8000 rpm for 10 min each time. Finally, BFO submicron cube powder was obtained by drying the reactants.BFO submicron cubes were synthesized by a simple hydrothermal method ,58, and GO was synthesized from natural graphite powder by a modified Hummer’s method, as mentioned in previous works ,60,61. B−2 . MB was chosen as the degradation material to examine the photodegradation of BFO and 3D BFO/graphene composites [In the photodegradation experiments, the CHI660E electrochemical workstation manufactured by Shanghai Chenhua Company was used for electrochemical performance tests. The test mode adopted a three-electrode system. The BFO/graphene composite-coated ITO-glass substrate, Pt wire, and Ag/AgCl electrode were used as the working electrode, counter electrode, and reference electrode, respectively. The light source in the photocatalysis experiment was a xenon lamp with a power of 150 W, and the parallel light power density of the xenon lamp was 70 mW cmmposites . Before The morphologies of the BFO and BFO/graphene composites were observed using scanning electron microscopy . The Raman analysis was performed on a Raman spectroscope (Renishawin Via Raman Microscope) with an argon-ion laser at an excitation wavelength of 514 nm. The crystal structures of the BFO and 3D BFO/graphene composites were characterized by X-ray diffraction . The surface areas of the BFO and BFO/graphene composites were estimated by measuring the nitrogen adsorption–desorption isotherms on a Micromeritics ASAP 2020 M micropore analysis system at 77 K. A spectrometer  was used to analyze the optical reflection of the samples and the maximum absorbance wavelength of residual MB dye after photodegradation experiments. The thermogravimetric analyses (TGA) of BFO and 3D BFO/graphene composites (B-3D3) were performed by a thermogravimetric analyzer  in an air atmosphere of 25–800 °C. The Total Organic Carbon (TOC) experiment was carried out by using a total organic carbon analyzer .θ of 33.11° indicates that BFO has the perovskite-type rhombohedra structure [2Fe4O9 and Bi2O3/Fe2O3. As shown in The quality of BFO at different hydrothermal times and pH values of the precursor can be readily observed from the XRD patterns with the same hydrothermal temperature (200 °C). In tructure . HoweverIn addition, the pH of the precursor affects the morphology of BFO. 2 hybrid carbon atom ring in graphene and the defects and disorder of the carbon lattice [It has been demonstrated that the single BFO submicron cube without the addition of GO has pure perovskite structure with good crystallinity. The red circle part in  lattice . In highAs mentioned above, the formation of pure phase BFO submicron cubes requires a strongly alkaline environment. The addition of GO destroys the alkaline growth environment of the BFO submicron cube, so when the concentration of GO is more than 3 mg/mL, the crystallinity of the BFO submicron cube is not good. However, when the concentration of GO is less, the BFO submicron cube tends to generate miscellaneous items. To sum up, the optimized concentration of GO is 3 mg/mL. 2/g for the BFO sample to 55.3 m2/g for BFO/graphene composites (3 mg/mL), indicating the porous structure of the composites.Furthermore, First, we studied the photocatalytic mechanism of BFO/graphene composites under visible light. A photocatalytic mechanism diagram is shown in αhv)2—hv curve shown in α (L/g·cm−1) is the absorption coefficient, h (eV·s) is the Planck constant, v (m−1) is the ratio of the speed of light with the wavelength, the hv value is 1240/wavelength, Eg (eV) is the band gap, and A is a constant.Therefore, we tested the optical absorption and obtained the band gap values of the 3D BFO/graphene composites under visible light. αhv)2 versus hv plot by extrapolating the linear portion of (αhv)2 to the energy (hv) axis at α = 0 [The band gap could be estimated from the (at α = 0 . Table 1We further tested the photocurrent change (P—T curves) of 3D BFO/graphene composites with different GO concentrations using a three-electrode system. The BFO/graphene composite-coated ITO conductive glass was used as the working electrode. The lighting condition was designed by adjusting the light switch with a time interval of 20 s. When lighting was added, the working electrode absorbed the light and produced a large number of electrons and holes. The electrons were exported through the ITO conductive glass, and the holes were trapped by the electrolyte to generate a current. In the absence of light, the light current was zero. Repeated cycles and good repeatability indicated that the physical and chemical stability of the photoanode prepared in this work is excellent. As it can be seen from − and h+ represent electrons and holes, respectively, which react with oxygen dissolved in water to form O2− and highly reactive radicals (⋅OH and ⋅OOH).Finally, we tested the degradation of MB dye by using the 3D BFO/graphene composites with different GO concentrations. We had five copies of each sample and did five experiments, and finally calculated the average value. The amount of catalyst was 0.05 g, and the concentration of MB solution (20 mL) was 40 mg/L. Before the photocatalytic tests, the 3D BFO/graphene composites were added to the solutions of MB dye for three hours to achieve the adsorption–desorption equilibrium. Since the characteristic absorption did not obviously change before and after the adsorption–desorption equilibrium, we suggest that the adsorption of MB dye on the BFO/graphene composites was negligible. The specific reaction process is as follows:In this paper, we prepared BFO submicron cubes by the hydrothermal method. It was found that the optimum conditions to form pure phase BFO submicron cubes with regular morphology are a hydrothermal time of 72 h and a precursor pH value of more than 14. Different 3D BFO/graphene composites were fabricated by adjusting the GO concentration. The results show that adding 3D graphene can adjust the band gaps of 3D BFO/graphene composites, thus greatly improving the photocatalytic activity of the composites. Moreover, better structure and photocatalytic performance of the 3D BFO/graphene composites can be obtained when the GO concentration is 3 mg/mL. Under visible light irradiation, the degradation rate of MB dye using these 3D BFO/graphene composites reaches 92% in 140 min. The enhancement of photocatalytic activity in visible light can be attributed to the large specific surface areas and three-dimensional architecture of composites, which can provide more transport paths and effectively improve the separation rate of photo-generated electrons and holes.


Graphene, graphene oxide, and reduced graphene oxide have been widely considered as promising candidates for industrial and biomedical applications due to their exceptionally high mechanical stiffness and strength, excellent electrical conductivity, high optical transparency, and good biocompatibility. In this article, we reviewed several techniques that are available for the synthesis of graphene-based nanomaterials, and discussed the biocompatibility and toxicity of such nanomaterials upon exposure to mammalian cells under in vitro and in vivo conditions. Various synthesis strategies have been developed for their fabrication, generating graphene nanomaterials with different chemical and physical properties. As such, their interactions with cells and organs are altered accordingly. Conflicting results relating biocompatibility and cytotoxicity induced by graphene nanomaterials have been reported in the literature. In particular, graphene nanomaterials that are used for in vitro cell culture and in vivo animal models may contain toxic chemical residuals, thereby interfering graphene-cell interactions and complicating interpretation of experimental results. Synthesized techniques, such as liquid phase exfoliation and wet chemical oxidation, often required toxic organic solvents, surfactants, strong acids, and oxidants for exfoliating graphite flakes. Those organic molecules and inorganic impurities that are retained in final graphene products can interact with biological cells and tissues, inducing toxicity or causing cell death eventually. The residual contaminants can cause a higher risk of graphene-induced toxicity in biological cells. This adverse effect may be partly responsible for the discrepancies between various studies in the literature. Nanotechnology is a multidisciplinary field of research spanning several diverse disciplines, such as biology, chemistry, engineering, materials, medical science, and physics. In particular, nanotechnology involves the creation and development of novel materials and devices through the manipulation of properties and functions of matter at the nanometer scale. At this length scale, the chemical, mechanical, physical, and biological behaviors of materials differ substantially from their bulk matter counterparts. Nanotechnology holds great promise for the development and synthesis of nanomaterials with unique chemical, biological, mechanical, and physical properties. By introducing nanotechnology in medical sector, scientists are capable of developing new materials and strategies for accurate detection and prevention of malignant diseases, novel medical implants and artificial prosthesis with good biocompatibility ,2,3.Carbon-based materials such as carbon nanotubes and graphene are widely known to possess exceptionally high Young’s modulus and mechanical strength, high light transmittance, and excellent electrical conductivity ,6,7,8,9.2 hybridized carbon atoms bonded covalently in a hexagonal lattice. It is a basic building block for carbon materials of all other dimensionalities, including 0D Bucky ball and graphene quantum dot (GQD), 1D carbon nanotube, and 3D graphite  at high temperatures . As a result, excess carbon domains are left behind on the wafer surface and are then reconstructed to produce graphene ,51. Howe2/Si, glass or flexible polymer such as poly (PET). The deposition and growth of graphene films takes place in two steps. The first step involves an initial pyrolysis of the precursor to form carbon. This is followed by the formation of graphitic structure from dissociated carbon atoms. In general, CVD growth of graphene on Cu substrate with low carbon solubility occurs through a surface adsorption mechanism. In the process, the carbon precursor decomposes and it adsorbs only on the metal surface, followed by migration and growth. In contrast, graphene on Ni takes place through carbon segregation and precipitation [2) on the meter-sized single-crystal Cu(111) foil through epitaxial growth of graphene islands on copper surface [2 V−1 s−1 at 4 K. High quality CVD-grown graphene films find attractive applications in making bendable touch panels, displays, and optoelectronic devices.Chemical vapor deposition (CVD) is typically employed to fabricate large-area, monolayer graphene films on transition metals  by admitting hydrocarbon gas, such as methane, ethane, or propane into the reaction chamber at high temperatures ,53. Cu opitation ,53,54. Cpitation . The gra surface . The as-2/Si, glass or PET is needed for practical applications. Such a graphene transferring process can induce defects, such as wrinkles and cracks, which are detrimental to its electrical and optical properties. To eliminate the transfer process, direct graphene synthesis on nonmetalic materials such as hexagonal boron nitride, without a need to transfer to another substrate has been explored. However, direct graphene growth is very slow and the final size of graphene film is also limited in the absence of transition metal Cu or Ni catalyst. Accordingly, the high cost of production and tedious film transferring process are the major obstacles for CVD-graphene commercialization at present [In general, direct transfer of graphene film from Cu or Ni foils to a target substrate such as SiO present . As ther present . Reduced present . Those LN,N-dimethyl-formamide (DMF); N-methyl-2-pyrrolidone (NMP); dichlorobenzene (DCB)) used are highly toxic; they can induce cellular toxicity; and, should be avoided for cell manipulation purposes.Liquid-phase exfoliation (LPE) is a scalable route for the mass production of graphene due to its low cost and simplicity. In the process, graphite is dispersed in a solvent to weaken the van der Waals forces between graphene interlayers in the presence or absence of surfactants. Ultrasonication or shearing can be employed to facilitate exfoliation of graphite into graphene sheets. This is followed by a purification step to yield single and multi-layered graphene sheets . This st2 and N2O4). To address these issues, Tour and coworkers modified this method by substituting sodium nitrate with phosphoric acid under a mixed H2SO4/H3PO4 (9:1) ratio while increasing KMnO4 concentration [4, and tedious sifting, filtration, centrifugation, and washing procedures. Therefore, several tactics have been developed to further modify the Hummers process, such as the employment of potassium ferrate (K2FeO4) as a strong oxidant instead of KMnO4 and the elimination of NaNO3 in the GO preparation [Graphene oxide is a graphene derivative produced by chemical oxidation of graphite flakes in strong oxidizing agents. Modified Hummers process is widely used to produce GO by reacting graphite flakes with a mixture of sulfuric acid, sodium nitrate, and potassium permanganate under vigorous stirring or sonication . The susntration . The advparation .GO is generally decorated with oxygen functional groups, such as carboxyl, hydroxyl, and epoxide ,34. Thus5 cm2 V−1 s−1 [Pure graphene exhibits exceptionally high elastic modulus of about 1 TPa and intrinsic strength of 130 GPa , superio V−1 s−1 . In this V−1 s−1 . The per V−1 s−1 . Polymer V−1 s−1 ,74,75,76As aforementioned, pristine CVD graphene is mainly developed for optoelectronic devices, touch screens, and solar photovoltaics. LPE-graphene flakes contain residual solvents and surfactants that have adverse health effects. In this respect, GOs with oxygenated groups are considered as ideal fillers for reinforcing polymers. Those functional groups render GO hydrophilic, thus can react with water-soluble polymer, such as polyvinyl alcohol . From thGraphene-based nanomaterials can be either biocompatible or toxic to biological cells. The response of living cells to these nanomaterials depends greatly on their layer number, lateral size, purity, dose, surface chemistry, and hydrophilicity. The surface chemistries of graphene nanomaterials vary greatly because of different strategies adopted for their synthesis, and the availability of different molecules or polymers for surface functionalization. Generally, several major cell lines are employed for in vitro evaluation of nanomaterial toxicity, including phagocytes  and non-phagocytic cells . Proper understanding of how graphene nanomaterials interact with those cells is of crucial importance in using them for medical applications.The interactions of graphene nanomaterials with cell membranes can also lead to the membrane damage and cytotoxicity. Phospholipids of cell membranes consist of a phosphate head group and two fatty acid chains. The primary head groups include choline, serine, glycerol, ethanolamine, inositol, and phosphatic acid. The various head groups in phospholipids confer them with distinctive properties. Furthermore, cell membranes also contain cholesterol molecules that play important roles in stabilizing membrane structure, maintaining fluidity, and modulating activities of membrane associated proteins. Pure graphene with no charges on the basal plane cannot interact electrostatically with phospholipids, but favoring hydrophobic interactions with the lipid tails. Moreover, hydrophobic interactions between pure graphene and cholesterol tail can extract or remove cholesterol molecules from the membrane, leading to the membrane damage. For instance, Bernabo et al. indicated that GO can interact with the cell membrane of swine spermatozoa, resulting in cholesterol extraction from the membrane . From thThe surface charge and surface chemistry of GO exert significant effects on the cellular interactions. GO has a high negative charge density conferred by oxygenated functional groups, thus possible electrostatic interactions between GO and lipids of the membrane may occur. In this context, Li et al. prepared five lipids with the same 18-carbon alkyl chain but different head group charges, and studied GO-lipid interactions using Langmuir monolayer technique . They foApart from the lipid membrane-nanomaterial interactions, graphene based nanomaterials can also enter cytoplasm because of their small size and sharp edges. They can penetrate cell membrane easily leading to membrane damage and leakage of cytoplasmic contents. Upon residing in living cells, they can induce toxicity through the creation of reactive oxygen species (ROS), which may cause mitochondrial disorder by reducing mitochondrial membrane potential (MMP), and cell membrane damage via the release of lactate dehydrogenase (LDH). In the latter case, ROS can induce lipid peroxidation by reacting with unsaturated fatty acids of membrane lipids to yield lipid peroxides, such as malondialdehyde (MDA). From these results, GO can induce extracellular and intracellular ROS generation, even at low doses, under a dose- and time-dependent manner. Therefore, ROS production, mitochondrial dysfunction and LDH leakage are the key factors relating to cell death as in the case of human HaCaT skin keratinocytes . If the The use of pristine graphene provides us with a better understanding of direct response and interaction between nanostructured graphene and biological cells. From literature, conflicting results have been reported relating the effects of pure graphene films on the biocompatibility and cytotoxicity of cellular systems ,93,94,952/Si. All graphene coated substrate specimens display a large increase in calcium deposits due to the bone formation. Furthermore, MTT assay results and immunofluorescence images reveal good cell viability and morphology. Kim et al. also demonstrated that CVD-grown graphene films exhibit stable cell attachment and excellent biocompatibility with primary adult cardiac cells (cardiomyocytes) [On the contrary, Nayak et al. found that CVD-grown graphene films do not hamper the proliferation of human mesenchymal stem cells (hMSCs), but accelerates their specific differentiation into bone cells . They inyocytes) . Graphenyocytes) .Cell migration and adhesion is coordinated by large protein complexes generally known as the focal adhesions (FAs). FAs contain high levels of vinculin, talin, paxillin, α-actinin, focal adhesion kinase, etc. The protein vinculin in particular controls the cell adhesion . It linkMore recently, Rastogi et al. studied the effect of LPCVD-grown graphene films on the viability and cell stress of both nonneuronal  and neuronal  cells . They reIn recent years, titanium and its alloys have increasingly been used for making dental implants. Ti-based alloys generally exhibit much higher corrosion resistance than stainless alloys ,98. HoweExtensive studies have been conducted on the biocompatibility/cytotoxicity of GOs due to their ease of fabrication and relatively low cost. GO can enhance cell viability and cause cell death depending on the size, dosage, time, cell type, and surface chemistry. Because of the different surface oxidation states and features between GO, rGO, and TRG, such graphene materials have distinct chemical and physical properties. GO possesses many defects, such as vacancies due to synthesis, as revealed by high-resolution TEM images and Raman spectra ,32. TRG 4) reducing agents led to higher Fe and Na concentrations in rGO. Fe and Na levels increased from 82 to 123 and 7600 ppm, respectively, by reducing with NaBH4. Residual Mn and Fe impurities in GOs affected their physical properties markedly [The synthesis of GO generally involves the use of several strong oxidizers that can introduce residual impurities or contaminants in the final products. Wong et al. determined the impurity types and contents in both GO and rGO. They reported that chemical oxidation and reduction steps during fabrication can introduce various metallic elements into GO and rGO . The maimarkedly .2+ activation through control of the rate of ATP synthesis during oxidative phosphorylation. As such, the presence of Mn in the nucleus of PC12 cells can lead to chromatin condensation, DNA fragmentation, and increased mRNA levels of caspase-3 [Heavy metals, such as Mn, Fe, and Cr are particularly harmful or toxic to mammalian cells. Manganese can induce toxicity in pulmonary and nervous cells ,103, andaspase-3 . Mn can aspase-3 . It is naspase-3 . In receaspase-3 ,106. Foraspase-3 . They rel-GO), 430 ± 300 nm (m-GO), and 160 ± 90 nm (s-GO) were used in their work. GO showed good biocompatibility with A549 cells and exhibited no signs of cytotoxicity , even at high GO concentration of 200 µg/mL. However, viability loss was observed for s-GO at 200 µg/mL. At this dosage, cell viability was 67% for 24 h incubation. Moreover, Trypan blue exclusion assay, in which the dead cells were dyed in blue and live cell remained unchanged was employed to assess cell mortality. Live cells generally exhibit intact cell membranes; trypan blue cannot pass through their membranes and enter the cytoplasm. On the contrary, the dye penetrates the porous cell membrane and it enters the cytoplasm of dead cells. Although s-GO induced viability loss at 200 µg/mL, no mortality increase of A549 cells is found. The mortality maintains at 1.5%, being similar to that of the control (1.4%)  . Using TFrom the literature, GOs exhibit dose- and size-dependent toxicity towards different cell lines, e.g., human fibroblast, human hepatocellular carcinoma, human skin keratinocyte, etc. ,47,87,88The effects of GOs on the biocompatibility/toxicity of immune cells are now considered. The interactions of GOs with the immune and circulatory systems are crucial; once administrated, they directly come in contact with such systems ,108. MacAs aforementioned, GOs can interact with cellular membranes and they induce damages through the formation of ROS, the extraction of membrane lipids or cholesterols, and the generation of carbon radicals. Red blood cell (erythrocyte) generally lacks repair capability due to the absence of a nucleus and mitochondria. The membrane damage of erythrocyte is termed as the hemolysis. Xia and coworkers indicated that lipid peroxidation could lead to a failure in the membrane integrity of murine erythrocytes. Hydrated GO induced severe hemolysis than GO and rGO . Liao etApart from the GO-erythrocyte interactions, little information is available in the literature relating the toxic effect of GOs on human primary cells. Primary cells are directly obtained from living tissue and established for growth in vitro. They mimic the physiological state of cells in vivo and produce more useful results representing human body system. Ethical permission for the acquisition of primary human cells from healthy and diseased tissue organs is needed. Very recently, Wu et al. assessed the toxicity of GOs upon exposure to primary human corneal epithelium cells (hCorECs) and human conjunctiva epithelium cells (hConECs) with doses of 12.5–100 µg/mL . From thGraphene oxides can be functionalized with a variety of molecules and biomolecules to increase their dispersibility in physiological solutions, thus improving their biocompatibility. GOs are typically functionalized with PEG, amine, acrylic acid, and dextran. In particular, GO-PEG has been shown to exhibit good biocompatibility as compared to GO ,114,115.2, poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA), and GO-PEG in vitro and in vivo [More recently, Xu et al. studied the viability of murine macrophages (J774A.1) treated with GO, GO-NH in vivo . Among t in vivo , rGO-PEG in vivo . The dos2S) for reducing GO. Sodium sulfide is a toxic agent that causes serious human skin irritation or burns, and it reacts with moist air or acid to produce toxic H2S. We considered that residual Na2S in rGO induces pro-inflammatory cytokines, leading to the activation of macrophages.Akhavan et al. prepared rGO nanoplatelets by sonication PEGylated GO sheets, followed by reducing in hydrazine and bovine serum albumin . The celTo address the cytotoxicity induced by hydrazine, Gurunathan et al. successfully prepared rGO using a bacteria strain, i.e., Pseudomonas aeruginosa as a reducing reagent for GO . They thMore recently, Dasgupta et al. used green synthesized rGOs of different dosages  for investigating their interactions with human lymphocytes derived from fresh blood samples . GOs werIn a recent study conducted by Mittal et al., GO was found to induce toxicity at higher levels than rGO upon exposure to human alveolar epithelial A549 and bronchial epithelial (BEAS-2B) cells . The preSasidharan et al. studied the effect of TRG and carboxyl-functionalized TRG (f-TRG) on the viability of monkey Vero kidney cells . Contacti.p.), intratracheal instillation, oropharyngeal aspiration, subcutaneous injection, etc. The in vivo effect of GO and its derivatives depends greatly on the chemical nature of nanomaterials, exposure time, dose and administration route, and type of the animals employed for the tests. From the early study by Yang et al., PEG-GO did not induce appreciable toxicity in mice after intravenous administration under a dose of 20 mg/kg for three months based on blood biochemistry and hematological and histological examinations [i.p. and oral administration followed by histological and hematological examinations [i.p. injection, its toxicity to the mice was insignificant. Ali-Boucetta et al. indicated that purified GO did not induce inflammation and granuloma formation in mice after i.p. injection [In the in vivo system, the whole living animal is employed to assess the effect of toxicity induced by graphene-based nanomaterials. Useful information relating cell-graphene interactions can be obtained by histological and microscopic examinations of relevant organ tissues and inflammatory cells of the animals after oral feeding, intravenous administration, intraperitoneal injection  with doses of 50, 150, or 500 mg/kg into Wistar rats [On the contrary, pristine LPE-graphene was found to be toxic and distributed mainly in the brain and kidney of chicken embryos . The finree days . They intar rats . GOs firtar rats . They re2, and GO-PAM at 0.05, 1, 5, 10, and 20 mg/kg body weight through intratail vein injection [2 and GO-PAA. GOs were mainly accumulated in the lung, spleen, and liver, as indicated by laser desorption/ionization mass spectrometry (LDIMS) analysis . From these, GO-PEG is moderately safer than GO in vivo, GO-PAA shows the best compatibility, and GO-PAM exhibits the highest cytotoxicty in vitro and in vivo.From in vitro cell cultivation, GO-PEG induced immune responses through cytokine secretion in murine macrophages. So, PEGylation did not help in passivating GO surfaces ,118. It njection . The suranalysis a. The feanalysis b. Histolanalysis c,d. In Hi.p. injection. High levels of ROS and MDA due to lipid peroxidation were responsible for the brain and kidney damages [i.p. administration, PrGO was first adsorbed in the peritoneal cavity and then accumulated in liver. Repeated injection of PrGO caused acute liver injury and increased splenocyte proliferation. The splenocyte proliferation stimulated immune system responses due to the presence of a variety of cell types with different immune functions, such as macrophages, dendritic cells, B cells and T cells inside the spleen. Similarly, Mendonca et al. reported that rGO and rGO-PEG weaken the BBB junction of Wistar mice as a result of ROS and lipid peroxidation generation [Syama et al. administrated PrGO (rGO-PEG) with a dosage of 10 mg/kg body weight into Swiss Albino mice through intraperitoneal and intravenous injections for 3, 7, 14, and 21 days . Confoca damages . Syama e damages . PrGO inneration ,137. Incneration . From thneration ,136,137.neration . Oxidatineration ,144,145.3 side groups in its repeated units. The methylene groups render PLA with a slow biodegradation rate, low mechanical strength, and toughness. Poor mechanical strength of PLA hinders its development for fabricating internal fixation devices for fracture bones in orthopedics. Biodegradable polymer fixation devices have a distinct advantage over their metallic counterparts because the devices can degrade in human body, so revision surgery to remove them is unnecessary. This greatly reduces the pain of patients suffering from bone fractures and the cost of hospital care. In recent years, the number of patients suffering from bone-related diseases, such as cancer and bone fracture, rises sharply due to an increase in older population and environmental pollution globally. In addition, sports and recreation activities also cause bone injuries in adults. There is an urgent need in biomedical sector to develop advanced biomaterials and polymer nanocomposites with good biocompatibility for bone tissue engineering applications. Over recent years, attention has focused on the improvement of biocompatibility and mechanical property of biodegradable polyesters that are reinforced with GOs [GOs can disperse well in water due to the presence of oxygenated groups. So, hydrolytic polymers, such as polyvinyl alcohol (PVA) and PEG, can react with GO readily in aqueous solution to form polymer nanocomposites . Hydrogewith GOs ,149. Thewith GOs ,151,152.Arriagada et al. studied the effects of GO (0.5–5 wt.%) and TRG additions (1–10 wt.%) on the biocompatibility of melt-mixed GO/PLA and PLA/TRG nanocomposites . The hum3+ ions) [Conventional hydrogels that were synthesized from using organic cross-linker have many drawbacks including poor absorption, mechanical and structural properties. Hydrogels are hydrophilic, three-dimensional networks of polymers that are capable of absorbing a large amount of water or biological fluids, thereby showing attractive applications in tissue engineering and drug delivery . GO is k3+ ions) . Very re3+ ions) .2/Si substrate is tedious. Nevertheless, high-quality CVD-graphene provides useful information relating its interactions with cells. Most in vitro cell culture studies revealed that CVD-graphene with a nanoscale dimension is biocompatible with several cell lines, especially enhancing fibroblast adhesion and promoting hMSCs differentiation into bone cells. These results are encouraging for potential applications of graphene in orthopedics. The preparation of LPE-graphene sheets requires toxic solvents and surfactants for graphite exfoliation. Those chemical reagents, especially the surfactants, are difficult to eliminate from final graphene sheets, thus the use of LPE-graphene in the biomedical field is limited. A typical example is the LPE-graphene induces cytotoxicity in the brain and kidney tissues of chicken embryos [In this review, we summarized and discussed the synthesis processes of graphene-based nanomaterials, their biocompatibility, and cytotoxicity. Furthermore, the in vitro and in vivo cytotoxicity of graphene-based nanomaterials were also addressed. The main challenge in graphene commercialization nowadays is to produce high quality graphene on a large scale and at low cost. The quality of graphene plays an important role in the biomedical field because the presence of impurities can have an adverse effect on mammalian cells by inducing cell membrane damage and apoptosis. Proper understanding of direct interactions between nanostructured graphene and mammalian cells can be obtained by using pure graphene. Although mechanical exfoliation of graphite flakes can produce pure graphene, the yield is very low for cell manipulation purposes. CVD-graphene is too expensive and its transferring process from copper or nickel foil to the target glass or SiO embryos .2S in rGO induces serious pro-inflammatory responses in macrophages [So far, GOs prepared by the modified Hummers process are widely used for investigating their interactions and responses with mammalian cells in vitro and in vivo. Conflicting results are found in the literature about the biocompatibility and cytoxicity of GOs and rGOs under in vitro and in vivo conditions. This is partly related to the researchers employing various oxidation times, different types, and different concentrations of oxidants for the synthesis, producing GOs with structure and reactivity as well as impurity level that differ from one study to another. The diversity in the structural properties has a large impact on the cell-GO interactions. In addition, the various chemical oxidizers and reducing agents used to prepare GOs and rGOS can generate metallic impurities and organic contaminations, thus altering their interactions with cells, tissues and organs, and resulting in cellular damage and apoptosis. In particular, toxic hydrazine reducing agent causes significant cell destructions in hMSCs at very low rGO content (1.0 μg/mL) , while rrophages . These arophages .It is considered that the toxicity of GOs/rGOs can be minimized by dispersing them in polymers to form polymer nanocomposites. As such, graphene nanomaterials are immobilized since they are firmly bound to the polymer matrix of nanocomposites. In addition, the polymer matrix can seal the sharp edges of GOs/rGOs, preventing their penetration into cytoplasm. So, graphene nanofillers with large surface areas provide effective sites for cell adhesion and proliferation, especially for osteoblasts (bone cells). Furthermore, oxygenated groups of GOs can reduce the hydrophobicity of the polymeric matrix, thus facilitating cell attachment and spreading on the polymer surface as well ,16. This