Then, the key biomedical applications based on graphene materials are discussed, coupled with the current challenges and outlooks of this growing field. In a broader sense, this review provides a comprehensive discussion on the synthesis, characterization, functionalization, evaluation, and application of p‐G, GO, and rGO in the biomedical field, highlighting their recent advances and potential.Since its discovery in 2004, graphene is increasingly applied in various fields owing to its unique properties. Graphene application in the biomedical domain is promising and intriguing as an emerging 2D material with a high surface area, good mechanical properties, and unrivalled electronic and physical properties. This review summarizes six typical synthesis methods to fabricate pristine graphene (p‐G), graphene oxide (GO), and reduced graphene oxide (rGO), followed by characterization techniques to examine the obtained graphene materials. As bare graphene is generally undesirable in vivo and in vitro, functionalization methods to reduce toxicity, increase biocompatibility, and provide more functionalities are demonstrated. Subsequently, in vivo and in vitro behaviors of various bare and functionalized graphene materials are discussed to evaluate the functionalization effects. Reasonable control of dose (<20 mg kg This article is a comprehensive review on the synthesis, characterization, functionalization, toxicity/biocompatibility, and potential of graphene materials in biomedical application. It critically discusses the typical synthesis methods to fabricate graphene materials suited for biomedical applications, followed by a range of structural and functional characterization techniques to characterize the obtained graphene materials. The challenges, potential solutions, and perspectives are also discussed. Naked graphene materials, including pristine graphene (p‐G), graphene oxide (GO), and reduced graphene oxide (rGO), were reported to obtain unique properties such as large surface area, high absorption of laser, high elasticity, good charge‐transfer ability, and ferromagnetic properties. Thus, graphene materials have gained wide popularity in biomedical applications, including medical imaging, drug/gene delivery, photothermal therapy, tissue engineering, biosensing, and immunotherapy. However, the toxicity of naked graphene materials and their poor biocompatibility impede related research. To solve this problem, both covalent and noncovalent functionalization methods are utilized to not only reduce toxicity and improve biocompatibility by using polymers such as poly(ethylene glycol) (PEG), poly (PVA) but also to functionalize graphene‐based materials with various biomolecules. Though the application of graphene materials in the biomedical sector has been developing rapidly in recent years, several challenges remain to be addressed before their commercialization for biomedical use.Graphene is a single or few atoms thick sheet of space area,1 high functionalization methods, toxicity, and their applications in the biomedical sector, particularly in biomedical imaging, and drug delivery. This review expands on the previously published work and provides a comprehensive overview of the life cycle of graphene materials, comprising the synthesis processes, characterization and analysis, functionalization methods, toxicity and biocompatibility evaluations, as well as the applications of p‐G, GO, and rGO in various biomedical areas.Existing published reviews on graphene and its derivatives generally include individual synthesis strategies,18 funFigure The whole structure and content of this review is demonstrated in 2Various synthesis methods have been employed to produce graphene materials with different properties. Understanding the synthesis methods, postprocessing (dispersion), and characterization techniques would benefit medical and biological scientists when choosing suitable graphene materials from an initial stage during the exploration of biomedical applications. This section provides an overview of the preparation of graphene for biomedical applications from the aspects of synthesis, dispersion, and characterization. Typical approaches to fabricating graphene for biomedical applications are involved in the first part, including top‐down and bottom‐up methods. After synthesizing graphene, the graphene materials are generally dispersed or separated in a polar solvent to obtain a uniform distribution, which benefits further functionalization and provides a uniform dose during applications. Characterization techniques are categorized into structural and functional analyses and can be applied to observe and evaluate the prepared graphene‐based materials.2.1 especially scalable production, classification, and application of graphene have been thoroughly reviewed. In targeting biomedical applications, large‐scale synthesis of graphene with high purity, easy dispersion, and functionalization is preferred. Therefore, synthesis methods focusing on fabricating graphene with more than 200 mg or films larger than 200 cm2 and generating little harmful impurities are discussed in this section. This section provides an overview of the six approaches to fabricating graphene for biomedical applications, where top‐down methods include mechanical exfoliation, chemical exfoliation, reduction of graphene oxide, and bottom‐up methods involving chemical vapor deposition, arc discharge, and flash heating are introduced.Synthesis methods have been comprehensively reviewed and summarized from various dimensions,23 esp2.1.1 The improving trend in the last decade of mechanical exfoliation could be summarized as from small‐scale repeated scotch peeling to large‐scale microwave, ultrasound, or shear mixing assisted mechanical exfoliation. Recent advancements have applied these mechanically assisted exfoliation methods to synthesize graphene as raw materials in large‐scale for biomedical applications. For instance, Pang's group described an extrinsic corrugation‐assisted mechanical exfoliation (ECAME) synthesizing monolayer graphene on SiO2 substrate. With thermal treatment, the graphene–SiO2 adhesion energy overcomes the graphene layers; under such an energy‐dependent manner, monolayer graphene could be fractured after removing the top graphite flake with scotch tape. The ECAME method allows large‐scale single‐layer graphene (SLG) production that exhibits great potential as a key material for biosensors after further functionalization. Similarly, Pirzado et al. investigated on mechanical exfoliation of pure graphite discs on a rough glass surface to obtain FLG. It is a high‐yield method with a simplified set‐up and fewer purification steps . Paton et al. first proposed a high shear mixing method for large‐scale generation of high‐quality graphene without adding surfactant or dispersant for intercalation, where the shear rate exceeded 104 s−1, and 100 g h−1 of graphene was generated using an equipment volume of 10 m3. Under centrifugal extrusion, liquid layer friction, and hydraulic‐induced forces, graphene was exfoliated from graphite, where graphene generation was many times more efficient than sonication, and it can be scaled up to an industrial level. Despite achieving relatively high productivity and quality, the exfoliated graphene was almost pristine (p‐G). Hydrophobic p‐G showed low biocompatibility and is relatively toxic in vivo, as discussed in Section  and lateral size before its utilization in the biomedical field.Mechanical exfoliation separates stacked layers by overcoming the van der Waals (vdW) forces through mechanical force.5 The 4) oxidant, alkali metal ions, dodecyl amine organic molecules, and supercritical CO2 are first used to increase the interlayer spacing of graphite. The graphene‐intercalated compounds are subsequently exfoliated into FLG sheets by heating, sonication or shear mixing in the second step. As oxidation via the introduction of oxygen atoms is the most common method to increase the interlayer spacing of graphite, chemical exfoliation generally produces GO and rGO. Betancur et al. utilized ammonium hydroxide as an intercalating agent and synthesized rGO directly from mineral graphite via chemical exfoliation. Gebreegziabher's group proposed a one‐step chemical route for synthesizing water‐soluble rGO at the thickness of 0.36 nm using sulfuric acid (H2SO4)/potassium permanganate (KMnO4) assisted by ultrasonication. The temperature during exfoliation also influences GO reduction, where GO was produced at 60 °C.Chemical exfoliation is a two‐step process where intercalating agents such as potassium permanganate , hydrophobin (HFBI), and dodecyl amine were reported to intercalate graphite via different pathways which related to the properties of molecules. Ionic organic molecules such as TOAB intercalate through electrochemical interactions under different reduction potentials, and hydrophobic HFBI interacts strongly with hydrophobic surfaces of graphite.In comparison, rGO was synthesized under the same conditions except at 120 °C.35 Alk Chemical exfoliation facilitates large‐scale graphene production, but dispersing graphene materials during intercalation may involve toxic solvents like N‐methylpyrrolidone (NMP), which are harmful to the environment and the human body. Additionally, the long processing time (on average 26 h) of chemical exfoliation is also a challenge to address for the efficient production of graphene materials. Wang et al. generated large‐scale graphene suitable for biomedical application, where they applied ultrasound (250 W) coupled with a shear mixer (4000 rpm) in supercritical CO2 with a reaction time of 180 min. Graphene was generated with a high yield (82.6%), and ≈60% of the graphene sheets were obtained in less than three layers. In this process, supercritical CO2, due to its low critical point and small molecular size, acts as an excellent interactive medium that assists graphene's chemical exfoliation with high efficiency and safety without any residue. Moreover, ultrasonication activated the edges of the graphite and enhanced the gap between layers while associated with the exfoliation of graphene from the gap based on shear mixing. Therefore, it is necessary to reduce the toxicity of the as‐produced graphene to the living body based on suitable intercalation molecules. Also, it is important to increase the efficiency based on assisted exfoliation processes such as ultrasonication, microwave heating or shear mixing to synthesize graphene materials targeted for biomedical applications.By contrast, polar organic molecules like dodecylamine intercalate through the host–guest chemistry, similar to alkali metal ions.12a Ch By contrast, rGO induces less membrane damage attributed to a low oxidation state, and few reactive surface groups are applied as raw graphene materials for biomedical applications. This method involves two steps: synthesizing GO from graphite, reducing GO, and producing rGO. Brodie's oxidation method, Staudenmaier method, Hofmann method, and Hummer's method were reported as oxidation methods, which generate toxic gases due to the application of strong oxidants, including fuming HNO3, concentrated H2SO4, and the process is also relatively time‐consuming. Modified Hummer's and rapid heating methods are more likely to be applied, considering the efficiency and hazards. Modified Hummer's method changes the ratio between NaNO3 and KMnO4, and the mixture is modified (H2SO4 to H2SO4/H3PO4 or H2CrO4/H2SO4) to conduct the reaction. Recently, several studies have applied rapid heating methods, where KMnO4 is excluded. The pure nitronium ion oxidation was combined with microwave heating to convert π‐conjugated aromatic structures and associated unique properties. Chemical, electric, and thermal reduction methods commonly reduce GO to obtain rGO. Applying chemical reductants that do not react with water is traditional. For example, hydrazine monohydrate, hydrazine hydrate, a large excess of NaBH4, phenylhydrazine, hydroxylamine, glucose, ascorbic acid, proteins, and novel green‐reductant like delphinium root extract were utilized as reductants. Electrochemical reduction is another reducing method, where rGO was demonstrated to precipitate on the graphite electrode with a voltage change from −0.8 to −1.5 V. This method is relatively clean and is based on the reaction of GO + H+ + e− = rGO + H2O. The third pathway is based on thermal reduction, either dispersing GO in an organic solvent like dimethylformamide, dimethyl sulfoxide, NMP, and then heating to the boiling point under reflux conditions or relatively clean and rapid photothermal and photochemical treatment that applies UV, Xenon flash, and KrF excimer laser. In conclusion, researchers tend to study methods that produce less residual gas and require less time on the first step of GO synthesis. Moreover, in the second reduction step, rGO produced differently shows various qualities. The products must be dispersible, biocompatible, and less toxic for biomedical applications. Therefore, toxic chemical reductants should not be involved in this process, and electron/laser‐related methods seem to be a good enhancer for saving time.GO reduction is applied to generate graphene materials, rGO, that possess medium oxygen‐containing functional groups between p‐G and GO, as p‐G and GO damage cells physically.38 By 2.1.2 by utilizing hydrocarbon or other carbon‐bearing precursors in the presence of a catalyst under various atmospheres. There are plenty of classifications of CVD based on the differences in the substrate, carbon source, pressure setting, and enhancing methods. Traditional CVD based on metal substrates has drawbacks as there would be loss of product, corrugation, contamination, and breakage of graphene samples during product collection from metal substrates. Graphene generated from CVD for biomedical applications is supposed to obtain a larger size and be free of transfer from substrates if applied as a biosensor; the adjustable layers and synthesis with lower temperature and less time would also be beneficial. Several modified CVD methods were proposed targeting these points, and it is noticed that existing CVD technology could achieve large‐scale production of high‐quality graphene sheets with a controllable number of layers. Chen's group demonstrated a near‐equilibrium CVD method that produces large‐scale graphene sheets on various dielectric substrates. Compared with metal substrates, due to the relatively higher energy barrier on dielectric substrates, the diffusion of carbon precursors on the dielectric surface does not affect the evolution of the graphene shape. Thus, the graphene is tested to be highly crystalline with clean, wrinkle‐free, breakage‐free morphology and obtain high carrier mobility (>5000 cm2 V −1 s −1). Results have also shown that with increasing growth time from 9 to 72 h, the lateral size of graphene increases up to 11 µm, indicating a large‐scale graphene production through this method. Tu's group proposed a CVD method based on Cu substrate to control the graphene layer synthesized by adjusting parameters including the reaction pressure, the flow ratio of CH4 and H2, the temperature, and the reaction time. In their results, graphene with 1–2 layers was produced with low pressure, multilayer (3–7) graphene was synthesized with atmosphere pressure, and the other three parameters were modified based on the applied pressures. Molecular dynamics simulation can probably predict suitable reaction parameters for the specific graphene layer instead of trial and error. Traditional CVD generally needs a high temperature (900–1000 °C). Several recent studies used plasma‐enhancing methods to produce graphene at a lower reaction temperature and reduce production costs. Compared with traditional CVD, this method provided a deposition time of less than 5 min and a lower growth temperature of 650 °C.Chemical vapor deposition (CVD) is widely reported in the synthesis of graphene materials, where the basic principle is to deposit graphene materials on different substrates56 by  With a direct‐current arc voltage applied across two graphite electrodes, the graphite in the anode evaporates, and graphene deposits on the cathode. The arc evaporation of graphite in the presence of hydrogen could produce graphene sheets with two or three layers and a lateral size of 100–200 nm. Studies have also explored other starting materials to synthesize B or N doped graphene. In the early stage, small‐size and low‐quality graphene flakes were produced through the arc‐discharge method under relatively high hydrogen pressure and without any catalyst. Therefore, modified methods changed H2 to CO2, NH3, or other mixtures to synthesize graphene or nitrogen‐doped graphene sheets more suitable for biomedical applications. The catalyst was found to affect the generated graphene layer, where Huang's group performed four experiments with different catalysts and carbon sources to investigate graphene productivity. It was reported that flake graphite produces gram‐scale and high‐quality few‐layer graphene sheets when catalyzed by zinc‐based catalysts (ZnO or ZnS). However, the graphene layers decreased when the experiment was repeated with Cu powder as a catalyst. As SiC was utilized as a carbon source, the synthesized graphene was mainly of one to three layers. However, the drawback of the arc discharge method is the nonuniform distribution of graphene; Borand et al. noticed that the deposits collected from the cathode and inner wall differed. They studied the formation mechanism and proposed that the interlayer spacing, crystallite size, number of average layers, and defect density varied with the position of the stainless‐steel reactor. Also, they showed that the interlayer spacing, crystallite size, and the average number of graphene layers collected from the anode region were larger than the front region. Graphene from the left‐side region of the reactor indicated the smallest value, while the defect density values exhibited an opposite tendency among the three regions. The formation may be influenced by the charge, and inert gas distribution in the reactor, perhaps further theoretical study of the charge distribution and heat and mass transformation during graphene formation is needed to solve the problem of nonuniform distribution of graphene during arc discharge synthesis; it would also be a solution that applies a sorting process like centrifugation based on the mass of graphene sheets to obtain uniform sized graphene.The arc discharge method generally applies pure graphite rods as anode and cathode, immersed in an inert gas like He.60 Wit Stanford et al. further explored the mechanisms and found that when high voltage is applied, the current travels along the path with the least resistance, thus providing more energy for the conducting area, thereby significantly annealing and graphitizing these areas. With the gradually increasing conductivity, the formation of conductive channels increases and a turbostratic graphene sheet is formed. The factors affecting graphene generation in FJH require further study before achieving high‐efficiency synthesis of specific graphene. Apart from the influence of carbon sources, heating duration is also important, according to Chen et al. Energy is increased with an increase in heating duration, and a phase change occurs from fluorinated amorphous carbon (≈10 ms) to fluorinated nanodiamonds (≈35 ms), fluorinated turbostratic graphene (≈100 ms), and fluorinated concentric carbon (≈500 ms). However, there is still no specific corresponding energy requirement for the formation of each structure; perhaps a molecular dynamics study based on the reactive force field could be beneficial to such studies. Another interesting study by Beckham et al. autonomously improved the crystallinity of flash graphene over many trials with machine learning based on the XGBoost regression model, where five features were chosen, and Raman spectral mapping was used as the training dataset. It is probably properties except crystallinity could be improved based on expanded ML or deep learning methods without manually choosing features if with a large enough dataset and well‐established neural networks.The flash heating method includes flash Joule heating (FJH) and laser radiation, where a rapid temperature increases, and cooling is involved. FJH, with its great potential, results in the high‐quality, large‐scale generation of graphene without applying solvents or energy‐consuming exfoliation. It was first reported by Luong et al., where the high voltage discharge from the capacitor bank allows the carbon source to reach temperatures above 3000 K in less than 100 ms, effectively converting amorphous carbon into turbostratic graphene.64 Sta Substrates of quartz and wood were applied in an early study; for instance, Ye et al. transformed wood into hierarchical porous laser‐induced graphene, where CO2 as a working substance induced laser with a wavelength of 10.6 µm. The laser obtained a maximum power of 75 W, and the graphene layer decreased with increased power. The laser‐induced graphene was found to be GO based on X‐ray photoelectron spectroscopy (XPS) characterization, attributed to the abundant oxygen‐containing functional groups in cellulose, hemicellulose, and lignin. Though the method is facile, and the size or layers of graphene are controllable, the laser‐induced graphene generated from wood etching required removal from the wood surface for further application. Thus, modification of the methods seems necessary for biomedical applications. Luong et al. proposed a one‐step infiltration process to solve this problem. The laser‐induced graphene was initially generated from polyimide film. In the infiltration process, hydrophobic materials such as polyethylene and wax infiltrated the laser‐induced graphene. After the polyimide film was removed, the laser‐induced graphene composites were synthesized. The application of such composite for thermal therapy was also tested in their study. Apart from the infiltration process for graphene transfer, it is probably uniformly distributed biomass powders with hundreds of micrometers of size radiated by high power laser in an inert gas atmosphere for a short duration that may also be a solution to generate transfer‐free graphene powder directly.In terms of laser‐induced graphene, sharply increased temperature from the laser breaks the chemical bonds and rearranges the carbon atom. Graphene materials are formed during the cooling process, where the reaction atmosphere, laser wavelength, light source, and starting material composition influence the graphene properties.68 Sub2.2 In terms of the dispersion mechanisms, graphene sheets aggregate based on vdW or π–π stacking; when there are interactions between solvents and graphene, the overlap of electron clouds could bring steric effects, causing tensions that overcome the interaction between sheets and pass the energy barrier from surface energy. Furthermore, graphene sheets are finely dispersed in the solvents. However, the performance of overlapped electron clouds induces steric effects, and the steric repulsion further induces tensions related to the property of solvents and graphene, specifically, the functional groups on the graphene surface and solvent particles. Dispersibility was reported to follow the trend: GO > rGO > p‐G due to the difference in the amount of oxygen‐containing functional groups on graphene; for instance, electrostatic repulsion of carboxylate groups brings the best dispersibility for GO. Additionally, the performance of solvents varied with the defects on the graphene surface, where water could be applied as solvents for rGO or GO, while p‐G was hard to be dispersed in water. However, specific solvents are favored under certain applications, and the dispersion efficiency is anticipated to increase. Therefore, dispersion assisting methods include microwave heating, sonication, shear mixing, and dispersants or surfactants. The application of surfactants is introduced in Section Dispersion of graphene in solvents must be achieved at a certain concentration and maintain stability for a reasonable period targeting the final applications.71 In π electron mobility in graphite or graphene. Then, the oxygen‐containing functional groups or solvent particles inside the layer soon decompose into volatiles, increasing the interlayer pressure and providing energy to disperse the graphene sheets. The power and irradiation duration influence the shape and concentration of dispersed graphene. Patel et al. synthesized microwave‐enabled low oxygen graphene for photoacoustic imaging, where microwave irradiation was applied for 30 s at 300 W. The graphene concentration in water was 0.4 mg mL−1 and kept undisturbed for five days. Tak et al. applied higher energy (900 W) for a longer time (5–10 min), where graphene quantum dots instead of graphene sheets were obtained, which may be because of higher energy decomposing the sheets. They provided the concentration in mm (16 mm mL−1); if converted into mg, it is ≈11 mg mL−1. Gürünlü et al. applied a one‐pot synthesis of GO sheets based on 100 mg mL−1 graphite solution assisted with microwave heating , and an optimum range for surface tension was determined at 45 mN m−1 according to their results. The irradiation duration and graphene concentration were positively correlated but require further confirmation. It was noticed that microwave heating was applied with a lower frequency than dispersion using mechanical methods such as shear mixing or ultrasonication, which may be attributed to heating that would induce the reduction of graphene sheets. Long‐term irradiation may lead to decreasing the sheet size.Low oxygen‐containing GO, and graphene quantum dots were dispersed based on microwave heating. For the mechanism of microwave heating enhanced dispersion, the microwave energy transforms into thermal energy due to the ive days.44 Tak However, the drawback is the requirement for longer sonication time. Several studies reported dispersion of graphene materials using PEG solution, where 1 mg mL−1 aqueous solution of PEG‐6000 and an aqueous suspension of rGO were sonicated for 30 min at room temperature. In another study, 2 h sonication was applied for GO in an ice bath, and a concentration of 0.5 mg mL−1 was obtained. GO dispersion reported by other researchers presented a similar experimental setting of sonication duration. A study of synthesis p‐G‐poly microbubble applied 90 min ultrasonication (320 W) in an aqueous solution of glycolic acid ethoxylate‐4‐nonyphenyl ether, and the composite was stable at a concentration of 0.25 mg mL−1. It was interesting to find that though GO obtained better dispersity, longer sonication duration was applied for GO compared with rGO and p‐G; perhaps a study of optimal sonication time for graphene with different functional groups would be beneficial to increase the dispersion efficiency. Another drawback of sonication is the nonuniform distribution of graphene size after sonicating for a longer time, where a postprocess of centrifugation may be required to produce uniform graphene sheet size. Dispersion of graphene materials in polymer based on only sonication may not be effective when pretreatment of graphene materials is required. rGO could be a representative, and it may be unstable and reaggregate in a polymer matrix even with sonication during dispersion; thus, pretreatment of rGO is needed when targeting dispersed graphene in such a polymer matrix. For instance, rGO microsphere aerogels were dispersed better than rGO in polydimethylsiloxane (PDMS) composite with the assistance of ultrasonication, attributed to the transformation of rGO from a flat to a spherical shape. The composite was found to have great potential in wound dressing.p‐G, GO, and rGO were dispersed with the assistance of sonication based on the mechanism of the creation of shear stresses and cavitation in the solvent.71a Ho as both the concentration (8.6 mg mL−1) and graphene lateral size  were reported to be larger than obtained from sonication. In terms of the dispersion mechanism, the Taylor–Couette flow generated from the shear flow between a rotating inner cylinder and a concentric, fixed outer cylinder induces shear stress and separates graphene sheets. It is probably the Taylor–Couette flow in shear mixing that mainly applies shear stress for dispersion, which avoids inducing cavitation and is beneficial to bring less defect on graphene and retain the original lateral size. However, recent studies only reported dispersion of p‐G; the optimum shear rates and shear duration for dispersion of GO and rGO with different solvents may need further study.Shear mixing was proposed to be a more scalable route to disperse graphene compared with sonication,71a as2.3Various characterization techniques can be applied to observe and evaluate the synthesized graphene‐based materials. The characterization techniques are categorized into structural and functional analyses. Morphology , dispersion behaviors, and functional groups can be characterized by structural analysis techniques. Functional analyses can be applied to analyze intrinsic properties  and extrinsic properties of dynamic behaviors in the physiological environment. Section 2.3.1 thus, it is important to evaluate the layer and size of graphene materials. Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), scanning tunneling microscopy (STM) and atomic force microscopy (AFM) can be applied for the quantification of layers. Similarly, size can be evaluated based on SEM, TEM, and AFM. Defects in graphene include intrinsic defects  and extrinsic defects . Stone–Wales defects benefit from increasing the electronic conductivity of graphene when working as biosensors, while extrinsic defects could work as sites for functionalization; thus, defects are not undesirable in graphene for specific biomedical applications. STM, TEM, AFM, and Raman microscopy can be used to detect defects.The layer and size of graphene determine the behavior of graphene materials for drug or gene delivery in biomedical applications. Under the same concentration, fewer layers or larger sizes bring a larger surface area for carrying drugs or genes. Additionally, the toxicity and biocompatibility of graphene materials were reported to be influenced by the size;79 thu The Raman scattering involves three steps: 1) photon excites electron vertical transition, resulting in electron–hole pair, 2) electrons or holes are scattered by phonons, and 3) scattered electron–hole recombination produces scattered photons. Considering the conversion of momentum and energy during phonon dispersion of graphene in the high symmetry ГM and ГK directions of reciprocal space, six phonon dispersion curves could be derived and applied in calculating the Raman spectrum  appears at 2700 cm−1 induced by stacking orders (second‐order dispersive Raman feature). The defects in graphene can be quantified with ID/IG. Increasing ID/IG demonstrates higher defect density for regions with low defect density. For regions with a high defect density of amorphous carbon structures, decreasing ID/IG corresponds to higher defect density. The reduction effect can be studied based on ID/IG, which increases with an increased degree of reduction; for instance, GO = 0.96, partially reduced GO = 1.12, and rGO = 1.6 have been reported. As Raman spectroscopy has accuracy limitations, Sandro's group developed a tip‐enhanced Raman spectroscopy (TERS) to overcome the diffraction limit of confocal Raman spectroscopy. According to their results, TERS could detect individual point defects with higher resolution. This could be a potential detection method for detecting the point defects of functionalized graphene for biomedical applications. In terms of the thickness measurement, generally, monolayer graphene shows a lower G band intensity, whilst bilayer or multilayer graphene possesses a much wider and upshifted 2D band than monolayer graphene. Though providing nondestructive imaging for graphene, the thickness measurement by Raman spectrum is limited, where more accurate results are obtained from high‐quality graphene with few layers (N < 4) compared with defective or thick graphene sheets. As defective or thick graphene would also be applied in the biomedical field, other techniques to measure thickness are introduced in the following paragraphs.Raman spectroscopy could measure the layer of graphene and detect the defects. The basic principle of Raman spectroscopy detection is based on the lattice vibration of phonon.31 Them Figure.[83] based on the discretely distributed secondary electron intensity, where the contrast in images represents thickness difference. The primary electron acceleration voltage (Vacc) is a crucial parameter in SEM; with high Vaac, fewer secondary electrons emit from the graphene flakes than from the SiO2 substrate; thus, the intensity contrast is small  if tapping mode AFM imaging with free amplitude of cantilever oscillation that is directly affected by nanomaterial–tip interactions without optimization. Shearer et al. proposed an accurate PeakForce tapping (PFT) mode AFM imaging to measure the thickness of graphene based on the hypothesis that pressure (peak force) determines the accuracy of measurement. With the PFT mode, the measure of SLG thickness was found to be accurate (0.43 nm) when increasing the peak force from 1 to 10 nN. As AFM may cause damage to the crystal lattice during measurement, it is probably that the PFT mode AFM imaging would be more convenient and cause less damage if the interface with a machine learning algorithm trained from interaction force, adhesion and topography dataset, and automatically determine the suitable force applied for regions in graphene sheets with variation in thickness.AFM can be applied to measure the lateral size and thickness and detect defects. The imaging mechanism of AFM is based on detecting variations in the reflected laser induced by the cantilever movements when scanning the samples, where the cantilever movements are affected by the vdW between the pin and sample. AFM takes advantage of high resolution, the magnification of an optical microscope is generally not more than 1000 times; the magnification limit of an electron microscope is 1 million times, while the magnification of AFM can be up to 1 billion times.18 As  as the infrared absorption spectrum is a typical molecular absorption spectrum, and the types of graphene materials can be distinguished based on the bond type and absorbance. When the sample is irradiated by infrared light with continuous frequency changes, the molecule absorbs energy from radiation with a specific frequency. Its vibration or rotational motion induces a net change of dipole moment. This further results in the transition of molecular vibration and rotational energy level from the ground state to the excited state, weakening the transmitted light intensity corresponding to these absorption regions. The FT‐IR spectrum is obtained by recording the curve between the percentage transmittance of infrared light and wavenumber or wavelength after Fourier transform. Some corresponding standard relationships exist between wavelengths in the FT‐IR spectrum and bond types. For instance, 1620 nm is attributed to the aromatic C=C bond, 1060 nm corresponds to the C—O stretching vibration mode in the alkoxy group, 1170 nm originates from the epoxy C—O stretching peak, 1400 nm arises from the C—OH carboxyl group, 1740 nm is attributed to the C=O stretching mode in the carboxyl group, 2930 nm is assigned to C—H, and 3420 nm is for the O—H groups , ultraviolet–visible (UV–vis), XPS, and X‐ray diffraction (XRD) are typical characterization techniques for functional group detection of graphene. FT‐IR is widely utilized to analyze the bond types in molecules1 as ts Figure . Electrons excited by photons are called photoelectrons, and the intensity (or counts) or corresponding energy of these photoelectrons are needed to derive an XPS spectrum. Usually, peak positions in XPS are related to different functional groups. The functional groups in the samples can be ensured to identify further the type of graphene materials  exhibits no change. By contrast, the intensity of carbonyl  peaks significantly decreases in the case of rGO.XPS radiates samples with X‐rays so that the inner or valence electrons of atoms or molecules are excited and ejected.1 Elecs Figure .[50] According to the Bragg equation, 2hkldsinθ = nλ, where nkld is the grating constant, is inversely proportional to θ; the larger the crystal plane spacing, the smaller the diffraction angle. Each crystalline substance has specific structural parameters, including lattice type, cell size, etc. Thus, the number, position, and intensity of the diffraction peaks are characteristic of each substance, like a person's fingerprint. Although there are thousands of substances, there are almost no two substances whose diffraction spectra are the same, so a qualitative analysis of the phase of the substance can be carried out. The XRD spectra of GO and rGO showed in Figure nkl in GO due to the oxygen moieties between graphene layers. Thus, the peak at 12.2° of GOs shifts to 26.64° of the rGO, which brings an obvious difference in the XRD spectra between the two samples.XRD utilizes diffraction and scattering of X‐ray photons during irradiation to derive information, including crystal shapes, phase, and functional groups of samples.18 Acc The graphene concentration could be calculated with Lambert–Beer law (A = l × α × C) from the UV–vis spectrum, where A denotes UV absorption in graphene, l is the optical path length in cm, α represents the absorption coefficient, and C is the concentration of graphene. It is noteworthy that α depends on the lateral size, layers, and the number of functional groups of graphene; thus, it needs to be modified when applied to examine different samples. It could be determined either from the gradient of an absorbance/length versus concentration plot or using the literature values.Dispersion behaviors include concentration, stability, and decomposition/aggregation of graphene materials in solvents, which influence the functionalization and treatment efficiency, biodegradation, and transportation of graphene‐based products. UV–vis is generally applied to analyze the concentration; stability can be measured by Zeta potential, while decomposition or aggregation of graphene materials can be evaluated by dynamic light scattering (DLS). In the last part, UV–vis is introduced to detect functional groups; while increasing UV wavelength and achieving a maximum wavelength >500 nm, the adsorption curve would be flat, and optical density is dependent on concentration.71a Th For instance, graphene in water is typically negatively charged on the surface, and the close interface layer would obtain opposite‐charged ions. There exists a diffusion layer outer the interface layer, where solvent particles and dissolved ions move in this layer. Furthermore, a shear plane in this layer is chosen, ions inside the boundary move with the particle, but ions outside the boundary do not move with the particle. The potential at the shear plane is defined as Zeta potential, where the value generally <10 mV demonstrates unstable, 10–30 mV indicates slightly to moderately stable, 30–60 mV represents good stability, and samples with the Zeta potential >60 mV exhibit excellent stability. It is noteworthy that long‐term stability is important for the commercial application of graphene solvents; thus, a routine Zeta potential measurement is necessary for long‐term evaluation.The stability of graphene materials is evaluated based on Zeta potential in solvents. When graphene is immersed in a solvent, as the solid surface generally obtains a certain surface charge density, and the liquid has dissolved ions and a high dielectric constant, the interface will be charged.74 For The basic principle of DLS for size measurement is monitoring the temporal change in scattering intensity as the particles move under Brownian motion. It is noteworthy that the lateral and rotational diffusion parameters need to be considered when dealing with the raw data, as graphene sheets are generally flake instead of spherical particles. For instance, the lateral size of GO was determined from the translational diffusion coefficient with depolarized DLS using a model for circular, infinitely thin disks. The radius of GO was measured as 157 ± 22 nm based on the spherical model, which is smaller than the real value, while the radius provided with their model was more accurate (194 nm). Therefore, DLS with a suitable model would be more accurate and beneficial to the long‐term evaluation of biodegradation behavior.DLS is generally applied to measure the size of graphene sheets in solvents to evaluate whether they are decomposed or aggregated to smaller or larger clusters during dispersion.46 The2.3.2 The shape of CV reflects the detection speed, intensity, and reproductivity of biomolecules and evaluates the working life of graphene‐based biosensors. Sensogram could provide information on resistance variation with an increase in dose on the biosensor, demonstrating the sensitivity and limit of detection for the graphene‐based biosensor. Laser absorption could be evaluated by observing the temperature rises induced by the NIR laser. For instance, an 808 nm NIR was applied on GO and rGO solvents, and the temperature rise varied with time, demonstrating the photothermal therapy efficiency of graphene materials and assisting with determining suitable irradiation duration during therapy. Mechanical properties like stress, strain, and Young's modulus could be analyzed by the Zwick machine or AFM.The synthesized graphene materials must be applied in biomedical fields; therefore, electrical conductivity for biosensing, laser absorption for photothermal therapy (PTT), mechanical properties for tissue engineering, ferromagnetic property for magnetic field drive, and thermal stability of graphene materials under different environments need to be well evaluated. The analysis of these properties is flexible and with wide alternatives; thus, several typical methods are introduced here. For electrical conductivity analysis, cyclic voltammograms (CV) measurements and resistivity changes on gate voltage were widely utilized.104 Th As there is a very weak force  between the atoms at the top probe of AFM and those on the sample's surface, indentation‐type AFM measurements can be utilized to analyze the mechanical properties. Prein et al. reported that the force required to push the tip into the sample is a quadratic function of the indentation depth. They presented cartilage's stiffness distribution at different timelines by providing a constant force and measuring the maximum hitting frequency  is generally applied to evaluate the thermal stability of materials35 and as side scattering is correlated to cellular granularity. Also, the secretion of cytokines induced by graphene can be detected by FCM. Pirone et al. developed a tomographic flow cytometry (TFC) method to directly demonstrate the intracellular biodistribution of graphene materials in 2021. After reconstruction of the tomography with different angles and times based on AM‐TFC algorithms, 3D graphene distribution in an NIH‐3T3 cell after 48 h treatment with GO has been presented , and GO was similar. However, GO (hydrophilic) circulated mainly as single flakes, whereas p‐G (hydrophobic) and f‐G  formed multiple large clusters in circulation.Observing the interaction of graphene in a physiological environment, especially the interaction with cells, is important in the biomedical application of graphene. Flow cytometry (FCM) can give quantitative measurements of the cellular uptake of graphene materials110 asd Figure . Another3 size‐, time‐related toxicity, poor dispersity, low biocompatibility, and poor biodegradation. To improve dispersion and stability, prolong circulation, and reduce cytotoxicity, basic functionalization based on noncovalent and covalent methods is applied by utilizing hydrophilic polymers or carboxylic acids. Successively, further functionalization using proteins, DNA, metal nanoparticles, etc., is useful for achieving a more specific application efficiently. There are differences between noncovalent and covalent methods; in terms of the basic principle, noncovalent approaches usually rely on the stabilization effects  of surfactants that adsorb at the graphene surface. By contrast, covalent bonds are formed when reactions occur based on the functional groups (—COOH/—NH2) between functionalized materials and graphene. In terms of features, noncovalent modification methods could preserve electrical conductivity and present a large graphene surface, while covalent methods provide better stability and robust mechanical properties. This section discusses the basic and further functionalization from both covalent and noncovalent methods, as shown in FigureBare graphene materials have drawbacks when applied in vivo, such as dose‐,43 siz3.1 and a hydrophobic surface, while bare GO or rGO obtain sharp edges and a negative charge surface. Also, bare graphene materials are synthesized with a few size or thickness control, thus causing dose‐, size‐, time‐related toxicity, poor dispersity, low biocompatibility, and poor biodegradation. Therefore, basic functionalization methods that utilize hydrophilic polymers or carboxylic acids to modify the properties of bare graphene are needed. After functionalization, the graphene materials obtain better dispersity and stability, less toxicity, and can support subsequent further functionalization with other molecules.Bare p‐G has few functional groups115 an3.1.1π–π stacking, electrostatic interactions, and van der Waals force. PEGylation of graphene is the most common and well‐established method. As reported by several groups, rGO tends to agglomerate irreversibly or even to restack into graphite through π–π stacking interactions. At the same time, the noncovalent bonding of PEG with rGO could largely improve the dispersity and stability and inhibit aggregation, which may be attributed to the increasing steric hindrance. The rGO toxicity was also pacified by PEGylation, with a concentration of less than 10 µg mL−1, and there was almost no toxicity without reactive oxygen species (ROS) generation. Additionally, amino‐terminated PEG could act as a bridge to link functional molecules by amido bond further.During noncovalent functionalization methods, mixing the materials at a suitable temperature and applying ultrasonication are needed. A large number of materials were utilized to noncovalently functionalize graphene via hydrogen bonding interactions,116π– and polystyrene sulfonate (PSS) to functionalize GO via π–π interactions. Natural polymer dextran could functionalize GO based on hydrogen bonding and π–π stacking. Moreover, PVA generally interacts with GO based on hydrogen bonding. Also, a few groups functionalized GO with cationic polymers like polyethyleneimine (PEI), polypropylene imine (PPI), and poly(amidoamine) (PAMAM) based on electrostatic interaction. Additionally, physical adsorption was applied in earlier studies; polyoxyethylene sorbitan laurate (TWEEN 20) was also physically adsorbed on rGO via the van der Waals force. Similarly, Fan et al. examined the functionalization of FLG with chitosan (a type of polysaccharide) in an acid solution to obtain better biocompatibility via physical adsorption.Apart from PEG, other hydrophilic polymers have been reported to improve dispersity and stability and reduce toxicity. Several groups utilized pyrene‐terminated side‐chain liquid crystalline polymers14 andNoncovalent functionalization methods require a relatively simple process where materials and graphene interact without a chemical reaction. Thus, the unique properties of graphene are preserved while dispersity, stability, and toxicity are modified. However, noncovalent interaction is not strong enough to undergo intense extrinsic stimuli like NIR laser irradiation or high‐temperature exfoliation. Also, noncovalent interaction cannot significantly change electronic transport properties if used in biosensing.1273.1.2 where carboxylic acids or hydrophilic polymers are widely used. Carboxylic acid such as tetraphenyl porphyrin bearing sulfonic acid groups or dilute nitric acid led to the attachment of carboxyl, epoxy, and hydroxyl side groups on graphene, with which the functionalized graphene is more dispersible in the aqueous phase. Although carboxylic acid treatment is relatively simple, this method may introduce other active molecules through bioconjugation chemistry or create more defects on graphene sheets due to oxidation. Therefore, hydrophilic polymers are more likely to be applied.Covalent functionalization methods are adapted to obtain a stronger interaction between graphene and other materials, as well as enhance electronic transport,127 wh Grafting‐from methods consider graphene the initiation site to grow a polymer from monomers. By contrast, in grafting‐to methods, the presynthesized polymers are end‐tethered to the graphene surface. For grafting‐from methods, various polymerization methods were utilized, including 1) atom transfer radical polymerization of polystyrene (PS), poly(tertbutyl acrylate),4 poly(methyl methacrylate) (PMMA), 2) condensation of poly(glycerol sebacate) (PGS) to graphene, 3) attaching PEI to GO based on ring‐opening polymerization, 4) the direct electrophilic substitution of polyether ketones to graphene, and 5) Ziegler Natta polymerization of polypropylene (PP) to GO based on catalysts. In terms of grafting‐to methods, different kinds of chemical reactions were used to provide strong covalent interactions, including 1) esterification/amidation reactions of carboxylic groups in GO/rGO with hydroxyl or amine groups in the polymers of PEG, PVA, and PEI, 2) nitrene cycloaddition for the functionalization of p‐G/rGO with single molecules of polymers like PS, PEG, 3) cross‐linking by amine‐induced ring‐opening/esterification/nucleophilic substitution to provide functionalized graphene/GO with polymers of epoxy or poly(N‐isopropylacrylamide), 4) opening of maleic rings in maleic acid, and 5) radical grafting of PMMA to rGO by phase transfer. It is not easy to precisely choose a suitable covalent functionalization method among manifold reactions. In TablePrevious research classified graphene modification methods with polymers or poly‐carboxylic acids as grafting‐from and grafting‐to methods.128 Gr3.2 nucleosides/nucleotides or metal ions. After functionalization, the graphene materials exhibited more unique properties and could be used in biomedical imaging, drug/gene delivery, tissue engineering, biosensing, and immunotherapy.Basic functionalization could only provide limited benefits for biomedical applications; thus, the successive further functionalization methods provide more functions to graphene materials by utilizing proteins,130 nu3.2.1π–π stacking, electrostatic and hydrophobic interactions, van der Waals forces, and interaction based on self‐assembly. The functionalization of graphene materials with proteins, doxorubicin (Dox), Pluronic F38 (F38), Tween 80 (T80), and maltodextrin (MD) generally involves hydrogen bonding and π–π stacking. The binding of DNA chains to GO sheets was found to occur through π–π stacking and hydrophobic interactions between the bases of DNA and graphitic domains of GO. Electrostatic/hydrogen bonding interactions were also found between the prime amines of bases and the oxygen‐containing groups of GO. Furthermore, the functionalization of several proteins utilized even more interactions. The adsorption of steroid hormones on GO nanosheets is a good example, where hydrophobic and electrostatic interactions present in long‐range up to 10 nm and short ranges (2–5 nm) interactions include π–π stacking, van der Waals forces, and hydrogen bonding. Several groups adapted self‐assembly to connect DNA, SS‐GrBP5 mutant, polyoxyethylene sorbitan laurate (TWEEN 20), and photoreceptor proteins to graphene.Similarly, noncovalent functionalization includes hydrogen bonding, proteins,15 doxIt is noticed that the noncovalent functionalization of proteins, nucleosides/nucleotides is always based on the synergistic effect of multiple interactions related to the distance between molecules and graphene. Noncovalent interaction is useful when the controlled release of drugs or DNA is needed. However, such connections are not strong enough to bind with the functionalization agents that need a stable connection. Hence, covalent interactions are introduced to provide stronger interactions.3.2.2 attaching BLUF proteins through the reactive N‐hydroxysuccinimide ester groups, and bonding enzymes, SBA‐15 antibody through —NH2 and reactive functional groups on graphene are reported. The covalent functionalization of proteins can support specific graphene‐complex targeting to target cells or tissues. For covalent bonding of graphene and small molecular compounds, reacting with thionyl chloride followed by the reaction with octadecylamine on graphene, chemical coupling of isothiocyanate groups and amine groups to link rhodamine B isothiocyanate, using carbodiimide‐catalyzed amide formation to bind Cypate are few examples. These small molecular compounds with a stable connection to graphene can be applied in PTT and fluorescence imaging. As for metal ions, the graphene sheet chelated metal ions, including Cu2+ and Gd(III), formed a complex system analogous to a metal compound with an expanded planar ligand. These complexes could be applied as imaging contrast agents or enhancing PTT. However, chemical reactions between graphene and nucleosides/nucleotides for covalent functionalization are not fully understood. Further covalent functionalization aims to provide more functions to graphene for specific biomedical applications. Generally, interactions between proteins, specific drugs, metal ions, and graphene sheets by this method are quite strong. Due to low concentration, these proteins, specific drugs, and metal ions induce negligible influence on graphene's basic properties.Stronger interactions provided by covalent functionalization are required if applied in PTT, biomedical imaging, or tissue engineering. Chemical reactions also induce covalent interactions. This part introduces the categories of interactions of proteins, small molecular compounds, and metal ions. In terms of connecting proteins, forming an isopeptide bond between graphene and SpyTag,57a at4 Bare graphene materials induce cytotoxicity in vitro and physiological damage in vivo, as reported by a few studies. Therefore, a clear understanding of the interaction mechanism between p‐G, GO, rGO, and various cells or biomolecules in vivo and in vitro is crucial to avoid undesired severe side effects during application.The toxicity and biocompatibility of graphene materials need to be carefully evaluated prior to any biomedical applications, and it would be optimal to conform to preclinical safety pharmacology guidelines of regulatory agencies such as the Food and Drug Administration (FDA), International Conference on Harmonization (ICH), and the European Medicines Agency (EMA).42 BarThis section introduces the interaction mechanisms, followed by five routes of cytotoxicity induced by graphene materials in vitro in Section 4.14.1.1 CKK8, and annexin V assay are generally used to measure cell viability, especially cytotoxicity induced by gene mutation tested by ingenuity pathway analysis (IPA). Considering various cytokine secretion, mRNA expression, and metabolites involved in the study, omics approaches combined with machine learning would be a high‐efficiency choice for analysis when carrying out all these three nanosafety investigations. In this section, the interactions between cells and graphene materials are first introduced. The five routes inducing cytotoxicity, including ROS generation, mitochondrial membrane potential (MMP) depletion, cell starvation, physical damages, and gene toxicity, have been interpreted.In vitro tests treat various cell lines with graphene materials outside the body to provide a basic idea of whether graphene materials cause severe damage to cells. MTT,122 CK Macrophages are vital in the phagocytosis of foreign bodies and in alerting the rest of the immune system against invaders to facilitate innate or adaptive immune responses. The bare graphene materials are first divided as p‐G, GO, and rGO, the uptake and clearance of macrophages for these materials are described, their side effects on macrophages are introduced, and finally, potential research areas that need further investigation are proposed.Before introducing cytotoxicity, it is important to understand the interaction between cells and graphene materials, where researchers widely utilized macrophages to study the interaction.140 MaFigure After 30 days, cells would gradually internalize p‐G. Relatively hydrophilic GO and rGO were reported to be nonspecifically captured by macrophages of the mononuclear phagocyte system in the liver and spleen and THP‐1 human acute monocytic leukaemia cells  and GO (hydrophilic), while a few focus on rGO. The potential contributing factor could be a large variation in the synthesized rGO. It is not easy to evaluate the degree of reduction in rGO quantitatively and the hydrophilicity of rGO ranges between p‐G and GO. Considering that the difference between GO and rGO has been noticed when treated on macrophages,143 itFive routes induce cytotoxicity, ROS generation, MMP depletion, cell starvation, physical damage, and gene toxicity. Generally, ROS generation is caused by two pathways in mammal cell lines, including 1) a defence mechanism against foreign substances by macrophages, 2) lipid peroxidation in the mitochondrial respiratory chain and polyunsaturated mitochondrial membrane. Generation of ROS in macrophages follows route (1), where ROS generation is part of a defence mechanism against foreign substances by macrophages as well as a marker for its activation, and the increase of ROS level activates signaling of various routes, finally induce cytokine secretion and cell apoptosis. p‐G was reported to activate mitogen‐activated protein kinase and transform growth factor‐β (TGF‐β) signaling, activating two proapoptotic members, Bim and Bax, of the Bcl‐2 protein family. Then, the activation of caspase 3 and its downstream effector proteins, such as PARP, occurs, and cell apoptosis is induced , lipid peroxidation, mitochondrial dysfunction, and eventually apoptosis or necrosis.145 Ged Figure .5.3.2 As the electromagnetic field is concentrated at the graphene edges, the IR beam can enhance the light interactions of attached protein molecules and present a plasmon resonance spectral shift. Different proteins obtained varied vibrational fingerprints; thus, proteins could be distinguished based on the plasmon resonance spectral shifts  in the presence of H2O2 and then produced a blue color. This catalytic reaction was dependent on pH, temperature, and H2O2 concentration. Their results showed that pH 4.0, 35 °C, and 150 mm were the optimal environments. Additionally, it was confirmed that the linear range of glucose detected was from 1 × 10−6 to 2 × 10−5 mol L−1, with a detection limit as low as 1 × 106 mol L−1. Though the catalysis of GO‐COOH and HRP were both ping‐pong mechanisms , GO‐COOH exhibited higher catalytic activity to TMB due to accelerated electron transfer.Rodrigo's group studied graphene's mid‐IR‐induced plasmon resonance to detect adsorbed proteins.174 Ass Figure . Accordi5.3.3 The graphene was prepared via CVD on Cu foil, then transferred to SU‐8 epoxy film (5 µm thickness) with PMMA supporting layer, followed by photolithography and oxygen plasma etching; finally, nitro and Ag were doped to improve the electrical conductivity. Compared with penetrating electronics that lack large‐scale, real‐time, and safe recording/stimulation, this graphene‐based electrode took advantage of low electronic noise, good biocompatibility, mechanical flexibility, and remarkable optical properties. Graphene with 1, 2, and 4 layers was tested, where increased layer provided lower impedance and higher capacitance, 4‐layer graphene obtained the best performance with ≈103 Ω with a frequency of 20 Hz and capacitance of 326.7 F g−1. In vivo tests with mice models demonstrated the efficiency of such electrodes, and it is supposed that this graphene‐based electrode could be used for potential applications in tinnitus, Parkinson's disease, Huntington's disease, depression, and schizophrenia treatment. In another study, two models of imaging were combined based on a graphene biosensor, where Kuzum et al. applied an Au‐doped graphene electrode achieving simultaneous electrical recording for interictal and ictal activity detection in population discharges and fast population spikes with durations less than 5 ms and calcium imaging using two‐photon microscopy based on an 840 nm femtosecond laser pulse to excite Oregon Green BAPTA‐1 AM (OGB‐1). The 50 µm electrode obtained a 5‐to‐6‐fold improvement in SNR and a 100‐fold decrease of electrical noise in electrophysiology. Due to the accessibility of the electrode to the brain, both good temporal and spatial resolution were brought. While the two‐photon microscopy was advantageous in providing less optical toxicity compared with confocal microscopy, the imaging depth was also improved according to the test of hippocampal slices from mouse brains. The in vivo multimode graphene‐based biosensors nowadays are mainly utilized.Some recent studies revealed the development of in vivo biosensors based on graphene materials for brain signals detection and electrical pulse delivery for treatment. Park et al. developed a graphene‐based electrical device that records and passes electrical signals into cortical areas for diagnosis and electrical stimulations embedded in a subset of the graphene multichannel array for suppressing epileptic seizures after detecting the epileptic signals.190 Th5.4 Most of the studies are related to tissue regeneration. Researchers aim to develop a method that overcomes the limitations of the lack of organ donors, disease transmission, and immune reactions using regenerated tissue produced with biocompatible scaffolds. Materials used for tissue engineering need to be biocompatible as well as without inducing immune reactions (not toxic). Graphene materials can be applied as substrates due to their unique electrical and mechanical properties. After a suitable functionalization, these substrates can mimic the nanometer dimensions of extracellular matrices (ECMs) and create predefined structures to assist the growth of cells. This section is divided based on the structural features, where substrates with porous, flat, or fibrous structures are discussed.Tissue engineering aims to control the proliferation and differentiation of cells to form artificial organs in vitro, which can provide substitutes for severely injured organs and present potential solutions for diseases with unknown mechanisms.1 Most5.4.1 Sarkar et al. investigated hydrogel for bone‐related biomedical engineering. They synthesized poly(acrylic acid) (PAA) hydrogel cross‐linked by GO to improve the toughness, extensibility, and stress distribution. It was noted that GO connected with PAA both via covalent cross‐linking  and physical cross‐linking  were responsible for achieving high toughness (7124 kJ m−3) and extensibility (elongation at break 4078%). However, in vitro cell tests for tissue regeneration were not conducted. Ma et al. synthesized nanocomposite sponges of sodium alginate (SA)–GO–PVA based on the freeze–thawing cyclic process and freeze‐dried molding for wound dressing application. With GO concentration at less than 2 wt%, the sponges obtained a homogeneously porous and interconnected network structure to offer proper water absorption, breathability, and mechanical properties beneficial for cell proliferation.Porous substrates synthesized with graphene and polymers take advantage of keeping moisture for cell proliferation, enhancing the transport of nutrients and proteins, and ensuring good mechanical properties.116 SaEscherichia coli and Staphylococcus aureus. Based on the hemolysis tests and CCK‐8 assay, they confirmed that the complex was nonhemolytic and could promote cell proliferation. Finally, according to in vivo tests, the SA–GO–PVA–NFX complex effectively prevented inflammation in the epithelial tissues, which successfully healed the wound and reduced scar formation. The complex could be gradually degraded  for its antibacterial property; in vitro tests demonstrated a gradual release of the loaded NFX effectively against ed Figure13a. Dep5.4.2 and differentiation, while single‐layer and small lateral‐size substrates can be applied in artificial organs to pacify organ transplant rejection. Stem cells could differentiate into cells with different characteristics based on suitable growth factors and tissue‐regenerative inducers. Graphene materials are found to take the place of growth factors and accelerate the differentiation of cells together with different tissue‐regenerative inducers. Based on different studies, the differentiation‐enhancement property of graphene was related to the usage of tissue‐regenerative inducers and the type of stem cells and substrate materials. Nayak et al. transferred graphene synthesized using the CVD method to substrates of PDMS, PET, glass slide, and Si/SiO2. Then hMSCs were cultured to observe whether hMSCs could differentiate into bones with the osteogenic medium that contained dexamethasone and lacked growth factors such as BMP‐2. Their results confirmed that the bone cells were detected with graphene‐based substrates, and the differentiation rate was similar to growth factors  cell lines were studied using graphene synthesized with CVD methods, and retinoic acid was utilized as a nerve inducer. Compared with glass substrates, N2a cells proliferate better on graphene transferred to Si/SiO2, and they observed enhanced differentiation into neuron‐like cells with graphene.Graphene showed good cell adhesion and charge transfer property, facilitating the activation of the intracellular signaling system. Thus, graphene could take the place of growth factors. However, if dexamethasone was provided on bare substrates without graphene, hMSCs differentiated into different cells at various bare substrates. Neurons were detected on uncoated PDMS, muscle cells were observed on PET, while stem cells on glass slides and Si/SiO inducer.9 Compd‐lysine (PDL), a specific cell adhesion factor, could improve the proliferation of N2a cells on any substrate. However, PDL exhibited better effects on graphene due to higher affinity, attributed to the differences in the arrangement between graphene and glass. The affinity of cells and substrates is supposed to be important in tissue engineering. The difference in affinity explains why graphene can take the place of growth factor, while cells on glass or Si/SiO2 are difficult to proliferate and differentiate. The different behaviors of stem cells are probably related to electron transfer, considering that the behaviors of cells are affected by membrane potentials. Different substrates provide varied electron transfer efficiency and, thus, different membrane potentials. Changes in membrane potentials may influence the connection between dexamethasone and DNA sites. When connecting at sites of enhancers, a specific RNA expression and cell differentiation may be induced, while silencer site connection may forbid the differentiation of cells.Additionally, poly‐tentials.58 Cha The zwitterionic citicoline (CDPC) was utilized to modify GO to improve dispersibility, provide adsorption sites for La3+ ions and improve the antibacterial property. They found that the complex exhibited good wound exudate absorption and moisture‐retaining abilities. According to in vitro tests, it illustrated good biocompatibility, and cells proliferated well without bacterial proliferation. Finally, the in vivo tests proved that the product healed wounds and formed a mature epidermal architecture in two weeks.Except for stem cells, proliferation in normal cells was also examined. Liu's group developed a PVA–GO–citicoline sodium–lanthanum (PVA/GO–CDPC–La) film for wound dressing based on the solution intercalation method.196 Th The PPy/GO with GO of 5 wt% was found to be nonuniformly coated on Ti, with a cauliflower‐like morphology, which benefited from providing larger surface roughness (208 nm) and wettability (26° water contact angle) for cell viability (94% in 7 days) in biomedical applications compared with those with 1 and 3 wt% GO. Adding GO was also proved to increase the ability against corrosion due to the creation of a reinforcement network in PPy that protect the Ti metal, where both pore and charge transfer resistance increased with switching potential from −0.55 to 1.25 V. Adhesion test demonstrated a less than 5% removal of the coatings for lattice edges (4B class). The immersion test verified that hydroxyapatite  could be grown on the PPy/GO–Ti composite. Their research demonstrated a potential direction of modifying and promoting the performance of Ti‐based on graphene materials for developing bone implants with enhanced stability, osseointegration and cell attachments. It is interesting to find a successful in vivo application of graphene materials as dental implants. Shin et al. proposed a method to do surface modification of the sandblasted, large‐grit, and acid‐etched (SLA) Ti disk (in vitro) and implant (in vivo) based on rGO. The roughness was increased while the contact angle of water decreased, which benefited the cell attachment and proliferation compared with the immobilization of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) on Ti.In vitro and in vivo tests of implants based on graphene materials were also reported in recent research, especially for bone and dental applications. Rikhari et al. developed polypyrrole (PPy)/GO composite coating on Ti implants. The GO was synthesized based on a modified Hummer's method; GO was then dispersed in aqueous oxalic acid and pyrrole solution with ultrasonication, and the composite coating was achieved by a one‐step electropolymerization.197 ThAdditionally, in vitro tests based on hMSCs demonstrated osteogenic differentiation, adhesion markers including alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and vinculin were enhanced by rGO without any osteogenic factors, probably due to the ability to absorb exogenous proteins. In vivo study implanted the SLA Ti‐rGO into a beagle dog's mandible. According to the results from the removal torque test, histomorphometric study, and µ‐CT images, the osseointegration and dental tissue regeneration were enhanced by the rGO group compared with others 8 weeks after the surgery. Their research is comprehensive, and promising and is believed to obtain great potential in applying dental or orthopaedic implants. The enzymes on substrates facilitated the cascading reactions of catalyzing ADP to AMP and AMP to adenosine, which reduced or reversed the platelet aggregation induced by ADP and suppressed thrombosis. Furthermore, adenosine produced in the reactions accelerated the endothelialization of TEBVs by controlling and optimizing the cellular energy metabolism and microenvironment without triggering inflammation. Even after losing the activity of the rGO–enzyme complex, the antithrombotic function of TEBV could be retained ) substrates for antiplatelet function and endothelialization ability of tissue‐engineered blood vessels (TEBVs).175 Thd Figure . Accordi5.4.3 The electrospinning process produced PCL/PGS nanofibers, and graphene with 1 wt% was added to generate the relative electrical conductivity necessary for the conduction of electrical impulses in cardiac tissue  (PCL)/PGS‐based nanofibrous scaffolds for cardiac tissue regeneration.107b Te Figure . Accordi Mohammed et al. did a comprehensive review of the antimicrobial mechanism and effectiveness of graphene‐based materials. Antibiotic‐resistant pathogens may lead to implants removal long‐term after surgery; However, metallic ions such as silver and nitric oxide (NO) were proposed to be antibacterial, and the efficiency and safety required to be improved as pathogens may inhibit ions penetration based on outer membrane modification or bind and pump out metals via efflux pump, additionally, the high dose may induce side effects such as irreversible pigmentation or inflammatory response. Therefore, graphene‐based materials were proposed to be applied due to their intrinsic antibacterial activity targeting bacteria, fungi, and viruses. The antibacterial mechanisms were mainly attributed to the five cytotoxicities induce routes, as discussed in Section  Lateral size, layers, particle shape, functional groups, and aggregation or dispersion of graphene materials were proposed as factors that influence antibacterial activity. Neither too large nor small size and thickness led to lower efficiency. It is suspected that the size and thickness factors induced different efficiencies in the routes that led to bacterial death. Thus, there would be an optimal size and thickness for specific bacteria determined by the surface energy of graphene materials, which requires further study based on experiments and molecular dynamics studies. Smooth‐top‐side and rough‐bottom‐side shape graphene obtained better antibacterial activity; however, the cytotoxicity of these materials for normal cells or systems containing both normal cells and bacteria was not mentioned in the tissue engineering studies. Aggregation of graphene and lower oxygen‐contained groups induced decreased efficiency in antibacterial ability, where decreased surface energy was the essence. Therefore, successful tissue engineering studies based on graphene materials, especially the long‐term and stable application, still require studies combining theoretical mechanisms and experimental phenomena.As bacterial infections are an obstacle to the long‐term and stable application of biomedical implants or artificial organs, all three categories of graphene‐based materials for tissue engineering require antibacterial properties if applied for in vivo or in vitro cultivation.199 Mo5.5 PTT is performed by delivering graphene materials specifically into the targeted nidus and utilizing the laser absorption and thermal conversion behavior of graphene materials to ablate the nidus under NIR irradiation. Moreover, damage to surrounding normal tissues should be negligible in an optimal PTT. According to recent studies, PTT techniques are classified as basic targeting PTT and enhanced PTT.Biocompatible graphene materials with high‐laser absorption and thermal conductivity are suitable for PTT.44 PTT5.5.1 The composite exhibited highly efficient tumor passive targeting and low retention in RES. The intravenous injection of 200 µL PEG–GO at 2 mg mL−1 (20 mg kg−1) to mice‐bearing tumors was tested, with the exposure of 808 nm NIR laser at a power density of 2 W cm−2 every two days, and the tumor ablated within one week without showing signs of regrowth in 40 days. Histology analysis demonstrated that 10–50 nm single‐ or double‐layered PET–GO would not cause significant toxicity to normal tissues. Also, mice were confirmed to remain healthy for three months after treatment  motif was attached to rGO–PEG (mean size of 18.8 nm). Their in vitro tests based on 15 W cm−2 NIR light for 8 min showed that U87MG cells were destroyed with 6.6 mg L−1 rGO–PEG–RGD, while control groups obtained ≈100% viability. Also, they proposed that rGO would be a lower‐cost NIR photothermal agent compared with Au nanoparticles and CNT.In the early stages of research, nidus‐targeting in PTT mainly relies on passive tumor targeting or attaching ligands to graphene materials without any control or enhancing strategies. Yang et al. covalently functionalized GO with PEG and conjugated Cy7 dye covalently to PET‐GO for labeling.2 The 5.5.2 As an organic NIR agent exhibiting near‐infrared fluorescence (NIRF), cyanine may produce fluorescence when treated with NIR laser via a radiative transition instead of a nonradiative transition, decreasing photothermal transmission efficiency. It is found that the NIRF would be quenched via FRET if cyanine is aggregated on GO, and the photothermal transmission efficiency can be promoted. Therefore, a pH‐dependent mechanism of the complex conformation was investigated to apply the FRET, where cyanine molecules tend to be dislocated from the PEG–GO surface at pH 8. The decrease in pH led to the aggregation of cyanine molecules on GO  in the liver and kidney after 24 h.The targeting and controllable PTT provide a more efficient treatment to focus sites than basic targeting PTT. The synergistic effect of combining PTT with other treatment techniques has also attracted much attention. Guo et al. synthesized pH‐responsive cyanine‐grafted PEG–GO for FRET‐enhanced PTT.8 As aO Figure . In thei−2, there were necrosis and regression of tumors after two days and without regrowth after 22 days. Hu et al. fabricated GO‐FA/γ‐CD/C60 nanohybrid for the synergistic therapy of PTT and PDT. They first functionalized GO with FA and bonded with γ‐Cyclodextrin (CD) via π–π interaction. Finally, the cavities on the GO‐FA/γ‐CD surface allowed C60 to be loaded based on host–guest chemistry  for 4 min, cell viability reduced to 16.2% even at a low concentration (30 µg mL−1) of this composite   Figure , facility Figure . They al5.6 2) prevention effects of nuclease enzymes based on steric protection of adsorbed nucleotide, and 3) small graphene layers could intercalate to DNA and induce DNA scission. Also, interaction energies between graphene and DNA nucleobases were reported as thymine (T) < cytosine (C) < adenine (A) < guanine (G) in aqueous solutions. Apart from basic interaction mechanisms, more focus was directed toward exploring applications. Gene delivery based on graphene materials could be applied for both the detection of diseases based on ssDNA and the gene therapy relying on plasmid DNA (pDNA) transfection or siRNA‐induced gene silence, where challenges from potential genotoxic effects of graphene materials to normal cells can be overcome by applying suitable functionalization methods. This section introduces gene delivery techniques from the diagnosis and therapy aspects.Apart from the delivery of drug molecules, graphene materials could also behave as carriers in gene delivery. The interactions between graphene materials and nucleic acids reported in previous research were mainly described in three unique modes 1) preferential adsorption of single‐strand DNA (ssDNA) over double‐strand DNA (dsDNA),1 2) p5.6.1 the stronger combination between ssDNA/MB and target DNA or RNA terminates the fluorescence quenching effects and reactivates fluorescence. However, the fluorescence signal is not strong enough to penetrate deep tissues. Thus, gene delivery for diagnosis is only available in vivo. Lu et al. proposed the protection of oligonucleotides from enzymatic cleavage during cellular delivery based on GO, ensuring the stability of target DNA detection. In their research, MB was adsorbed on GO (size < 100 nm) noncovalently, which not only protected MB from cleavage based on the steric hindrance effect but also enhanced cellular delivery due to high GO cell uptake  that brings a terminal‐labeled fluorophore can be applied in diagnosis. When the composites combine with the target DNA or RNA of nidus cells, viruses, and germs,176 th−1, cell viability was close to 100%. Xu's group utilized a self‐assembling strategy to synthesize multifunctional hydrogels from GO sheets and DNA. According to their results, due to strong π–π stacking, electrostatic and hydrophobic interactions between ssDNA and GO, the GO/DNA‐composite‐self‐assembled hydrogel (GO/DNA SH) exhibited good mechanical strength and high stability. The complex also exhibited a self‐healing property where dsDNA is re‐formed with excessive ssDNA in SH during the cooling process and self‐assembled to synthesize GO/DNA SH when heating recurred. The GO/DNA SH is also supported with loading dye for detection based on the electrostatic interaction between positively charged safranine O and negatively charged GO sheets. Sheng's group proposed an aptamer‐based sensing method based on PVP‐coated GO and a fluorescently tagged aptamer, similar to the aptamer biosensor discussed in Section  The complex obtained high sensitivity (10 fm) and an excellent detection limit of 5 ng mL−1 without affinity for the enantiomer of L‐VP. Compared with surface‐enhanced Raman spectroscopy and matrix‐assisted laser desorption/ionization mass spectrometry, this technique is more cost‐ and time‐effective when applied as a chiral selector.Additionally, the toxicity of this composite is negligible. Even with a concentration of 100 mg L and DNA.131 Ac5.6.2 The transfection effect of pDNA delivery is a typical example of gene therapy, where graphene materials functionalized with cationic polymers are utilized. Feng's group synthesized cationic GO–PEI (10 and 1.2 k molecular weight) with noncovalent electrostatic interaction, further bonded with anionic pDNA via a layer‐by‐layer assembly process for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. The cytotoxicity of GO–PEI‐10 k was lower than free PEI‐10 k but remained similar to EGFP transfection efficiency, while GO–PEI‐1.2 k obtained much better EGFP transfection than PEI‐1.2 k. They proposed that suitable molecular weight and N/P ratio were needed to reduce cytotoxicity and increase transfection efficiency. Recent investigations also considered the size control of GO and other additional functionalization methods. Teimouri et al. compared the transfection efficiency and cytotoxicity of GO conjugation with alkylated derivatives of cationic polymers, including PEI and PPI, and PAMAM. The three cationic polymers were connected to GO based on the surface carboxyl group, glycine, and spermidine. They further utilized a plasmid DNA of enhanced green fluorescent protein to evaluate the transfection efficiency and MTT assay for cytotoxicity evaluation. Compared with bare cationic polymers, the cytotoxicity of all three complexes decreased notably, while the transfection efficiency was significantly enhanced.The major obstacle in this area is developing a nonviral‐based, safe, and efficient gene delivery vehicle; in this regard, graphene could be a good choice.117 Th By the adsorption of oleic acid and then covalently linked PAMAM, the complex obtained good dispersity, biocompatibility, and high transfection efficiency. More recent research in this area also paid attention to synergistic effects. Kim et al. studied photothermally controlled gene delivery by conjugating BPEI and rGO via a hydrophilic PEG spacer. The PEG–BPEI–rGO complex attached pDNA via electrostatic interaction was confirmed to be stable in the physiological environment. They conducted an in vitro gene transfection study with PC‐3 and NIH/3T3 cells. It was demonstrated that the complex induced negligible cytotoxicity and enhanced gene transfection efficiency when treated with an NIR laser  weight ratios also affected the toxicity and transfection efficiency. Therefore, suitable functionalization methods and the C/P ratio need to be considered. Similarly, Liu's group synthesized GO–oleate–PAMAM hybrids for gene delivery.205 Byr Figure . Bafilom The GA–PEG–GO–dendrimer (GPGD) of ≈100 nm obtained good stability, low toxicity, high transfection efficiency, and induced negligible hemolysis according to an in vitro test based on HepG2 cells. They observed significant cell uptake of GPGD–antivascular endothelial growth factor A (VEGFa) siRNA by HepG2 cells and decreased expression of VEGFa in both mRNA and protein levels, which indicated gene silencing  with therapeutic functions could also be delivered. Qu et al. reported a system consisting of glycyrrhetinic acid (GA), GO–PEG, and polyamidoamine dendrimer (dendrimer) for siRNA delivery targeting liver cancer therapy.162 Thg Figure . In vivo5.7 and closely related natural killer (NK) cells are the main targets in immunotherapy. It has been noted that immune cell communication and activation were controlled by immunological synapses. The interactions of immunological synapses were based on clusters of cell surface molecules in size range of 10–400 nm. Previously, monoclonal antibodies like Rituximab or EB6 were utilized to activate B cells or NK cells. However, the activation was weak as the antibodies are unbound soluble reagents. Therefore, developing antibody nanoclusters based on acellular soluble reagents would be more efficient, and graphene materials with suitable sizes would be better.Immunotherapy revolutionized cancer treatments by transferring strategy from killing cancer cells directly to improving the natural immune defences. Leukocytes such as B and T cells11 and Biotin groups were linked to the —NH2 of GO–PEG, and then streptavidin was coated on the complex. α‐hCD16 as antibodies for CD 16 receptors on NK were finally attached to GO–PEG–biotin–streptavidin. According to their results, by controlling the size of nanoclusters to 150 nm, more than 50% of primary NK cells bonded with such complex and further induced cellular activation  for chemical therapy, and SB‐431542 (SB) was attached as a TGF‐β inhibitor. With intravenous administration of such complex in mice bearing 4T1 tumor, the local primary tumor was destroyed upon NIR irradiation, and metastatic cancer was inhibited  and experimental phenomenon . Future work should focus on studies of the interaction between graphene materials and different cell lines. In conclusion, the challenges need to be addressed soon. improving imaging speed, and reducing harm to the human body. Since graphene materials possess a large surface area, which may support multifunctionalization, a multimode imaging contrast agent could be developed based on graphene. For drug delivery, synergistic effects are more efficient than the simple delivery of drugs; thus, combining drug delivery with PTT immunotherapy would offer a better therapy. In biosensing and gene delivery for diagnosis, in vitro diagnosis dominates the market share. There is still not much research on in vivo diagnosis that achieves real‐time and dynamic evaluation, as the dissipation of fluorescence signals in deep tissue blocks the development of in vivo detection. Perhaps measurements based on spectrum shifts of mid‐NIR can be a potential solution. Applying medical cosmetology based on graphene materials functionalized with hyaluronic acid would be a potential research area for tissue engineering. Moreover, PTT would generally require furless conditions in the region of interest (ROI); thus, patients need to remove their hair if they have a brain tumor. Also, the bone penetration of laser would be another problem when treating brain tumors. Minimally invasive PAI applying fibers with micrometer‐diameter can combine biomedical imaging and PTT based on preadministrated graphene and suitable laser. Regarding the theoretical mechanism study of graphene interactions with cells, a deep learning/machine learning potential field that achieves highly accurate calculation comparable to ab initio molecular dynamics simulation and as fast as the potential empirical field is recommended. Though the training in the deep learning/machine learning potential field would be time‐consuming, an accurate study of the mechanism involves graphene materials entering into various cell membranes, graphene materials interaction with nucleotide, the surface energy of graphene materials with different sizes/thickness/functional groups, etc. would be beneficial for design experiments.The current scope of graphene materials in biomedical applications has been covered in many topics but still expanding. Several prospects are proposed for future work. Graphene materials have been developed and examined to function as contrast agents in enhancing imaging quality,25 impTo summarize, research in graphene materials for biomedical applications is still in its infancy but promising. They illustrate great potential if current challenges can be tackled and their unique properties can be properly and readily utilized.The authors declare no conflict of interest.


Aberrant proliferation of vascular smooth muscle cells (VSMCs) is the cause of neointima formation followed by vascular injury. Autophagy is involved in this pathological process, but its function is controversial. Recently, we found that methyltransferase like 3 (METTL3) inhibited VSMC proliferation by activating autophagosome formation. Moreover, we also demonstrated that METTL3 reduced the levels of phosphorylated mammalian target of rapamycin (p-mTOR) and cyclin dependent kinase 1 (p-CDK1/CDC2), which were critical for autophagy and proliferation regulation. However, whether mTOR and CDK1 mediated the function of METTL3 on autophagy and proliferation in VSMCs remains unknown.We showed that the activator of mTOR, MHY1485 largely reversed the effects of METTL3 overexpression on VSMC autophagy and proliferation. Rapamycin, the inhibitor of mTOR, obviously nullified the pro-proliferation effects of METTL3 knockdown by activating autophagy in VSMCs. Unexpectedly, mTOR did not contribute to the impacts of METTL3 on migration and phenotypic switching of VSMCs. On the other hand, by knockdown of CDK1 in VSMC with METTL3 deficiency, we demonstrated that CDK1 was involved in METTL3-regulated proliferation of VSMCs, but this effect was not mediated by autophagy.We concluded that mTOR but not CDK1 mediated the role of METTL3 on VSMC proliferation and autophagy.The online version contains supplementary material available at 10.1186/s13008-023-00096-5. Vascular smooth muscle cell (VSMC) proliferation is necessary for maintaining the structure of blood vascular system. However, both over-proliferation and insufficient proliferation of VSMCs have adverse effects on the body. For example, over-proliferation of VSMCs will lead to neointima formation, while insufficient proliferation will result in vascular structure disorder or even rupture. Autophagy was reported to be involved in VSMC proliferation and survival , 2. Howe. revealed that both autophagy initiation and mammalian target of rapamycin complex 1 (mTORC1) were repressed during mitosis of cancer cells, and cyclin dependent kinase 1  substituted for inhibited mTORC1 as the master regulator of autophagy during mitosis [Our recently findings demonstrated that methyltransferase 3 (METTL3), a major N6-adenosine-methyltransferase, inhibits the proliferation, migration and synthetic phenotype of VSMCs by positively regulating autophagosome formation . Whether mitosis . Interes mitosis . Both CD mitosis , 13. HowIn the present study, we found that mTOR activation largely abrogated the inhibitory effects of METTL3 overexpression on proliferation by suppressing autophagosome formation. On the contrary, mTOR inhibition nullified the pro-proliferation effects of METTL3 knockdown in VSMCs. However, neither mTOR activation nor inhibition has impact on the function of METTL3 on migration and phenotype switching of VSMCs. In addition, although METTL3 enhanced the phosphorylation of CDK1, CDK1 did not mediate the role of METTL3 on autophagy.Recently, we reported that METTL3 inhibited the proliferation, migration, and synthetic phenotype of VSMCs by activating autophagosome formation . We alsoOur results showed that in DMSO treated HASMCs, METTL3 overexpression noticeably inhibited the phosphorylation of mTOR, and MHY1485 significantly increased the level of p-mTOR in HASMCs with or without METTL3 overexpression  RNA methylation is found widely across species and participated in abundant of biological processes regulation, such as proliferation, apoptosis, pyroptosis and ferroptosis [6A was also found to involved in the VSMC differentiation, phenotypic switching, proliferation, and migration [6A RNA methylation [. revealed that METTL3 suppressed colorectal cancer cell proliferation and migration by negatively regulating p38/ERK pathways [6A modification [. demonstrated that METTL3 pretreatment accelerated cardiomyocyte proliferation and reduced cardiomyocyte apoptosis under hypoxic or myocardial infarction conditions [6A is widespread at over 25% of human transcripts, and different stimuli may cause m6A modification in different RNAs or different sites, leading to different outcomes.N6-Methyladenosine  were purchased from Hyclone. 0.25% trypsin–EDTA was obtained from Gibco. The penicillin–streptomycin (PS) was from Geview. The primers of METTL3 were synthesized by Sangon Biotech , as well as Dimethyl sulfoxide (DMSO). MolPure Plasmid Mini Kit (19001ES70), Polyethylenimine Linear (PEI) MW40000 (rapid lysis) (40816ES02) and 5 × SDS Protein Loading (20315ES) were from Yeasen . MHY1485 (mTOR activator) and rapamycin (mTOR inhibitor) were purchased from Selleck. RIPA-lysis buffer (#HY-K1001) was purchased from MedChemExpress. Pierce™ BCA Protein Assay Kit (23227) were from Thermo Fisher Scientific. Polyvinylidene fluoride (PVDF) membranes (IPVH00010) were purchased from Millipore. The antibodies used in this study including anti-Flag  obtained from Sigma; anti-β-actin  obtained from Abclonal; anti-p-CDK1, anti-p-mTOR , anti-mTOR , anti-ATG5 , anti-ATG7 , anti-p-ULK1  and anti-LC3  obtained from Cell Signaling Technology; anti-PCNA (1:1000) obtained from Genetex; anti-p-Histone H3 (1:200) is from Santa Cruz; anti-METTL3  and anti-CDK1  obtained from Proteintech. Other antibodies such as anti-α-SMA , anti-SM22α  are from Abcam. Secondary antibody (111–035-003) was purchased from Jackson ImmunoResearch Laboratories. Clarity Western ECL Substrate and Precision plus Protein Dual Color Standards were purchased from Bio-Rad Laboratories (USA). Transwell invasion chambers were obtained from Costar.Primary human aortic smooth muscle cells (HASMCs) were obtained from the ascending aorta when they underwent heart transplantation at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology and cultured as described previously , 40, 41.The human plasmids of METTL3 and knockdown were previously described by us , 20, 42.Western blot was performed as previously reported , 10. Bri4 cells) was seeded onto the upper chambers of a transwell culture plate and 800 μL of medium containing 10% FBS was added to the bottom chamber after adherence for 3 h. The plate was incubated at 37 °C and 5% CO2 for 12 h. The cells in lower chamber were fixed with 4% paraformaldehyde fixative for 15 min and stained with 0.1% crystal violet for 30 min, washed away gently with water. Unmigrated cells on the upper chamber surface of the well were mildly wiped away by wet cotton swabs. Finally, the cells were observed and photographed with a microscope.Transwell was used to detect the migration of HASMCs as previously reported , 11. Aft® Solution , and 4 μg of total mRNA was reverse transcription into cDNA by using Hifair® II 1st Strand cDNA Synthesis Kit (gDNA digester plus) . Then, PCR amplification was performed using Hifair® qPCR SYBR® Green Master Mix (No Rox)  in CFX connect™ real-time PCR detection system (Bio-Rad). The mRNA expression levels were normalized to 18S. Primers used in this study were as follow: ATG5 forward primer 5′-GCTTCGAGATGTGGTTTGG-3′ and ATG5 reverse primer 5′-CCATTTCAGTGGTGCCTTC-3′; ATG7 forward primer 5′-GCATCCAGAAGGGGGCTATG-3′ and ATG7 reverse primer 5′-GATCAAGAACCTGGTGAGGCA-3′; CDK1 forward primer 5′-CTGGGGTCAGCTCGTTACTC-3′and CDK1 reverse primer 5′-TCCACTTCTGGCCACACTTC-3′.Total mRNA was extracted with TRI ReagentThe data were analyzed by GraphPad Prism 8 software in this study. All the data were expressed as mean ± standard deviation (SD). The differences between the two groups were determined using the student t-test, and statistical differences between multiple groups were analyzed using a one-way analysis of variance (ANOVA). A p value less than 0.05 is considered statistically significant.Additional file 1: Figure S1. The mRNA levels of ATG5 and ATG7 were upregulated in HASMCs with METTL3 overexpression. (A) Quantification of mRNA levels of ATG5 in HASMCs with or without METTL3 overexpression. (B) Quantification of mRNA levels of ATG7 in HASMCs with or without METTL3 overexpression. *p < 0.05 versus lenti-Flag. Figure S2. CDK1 was significantly knockdown in VSMCs. (A-B) The protein level of CDK1 was evaluated by using western blotting in HASMCs with CDK1 knockdown or not. (B) Quantitative results of blots in A (n = 4). (C) Quantification of mRNA levels of CDK1 in HASMCs with or without CDK1 knockdown. * p < 0.05 versus lenti-shRNA.