Progressive fibrosing interstitial lung disease (PF-ILD) is a progressive phenotype of fibrosing ILDs with varying definitions and elusive clinical characteristics. We aimed to clarify the clinical features and prognosis of PF-ILD cases based on the deterioration of pulmonary function.Altogether, 91 consecutive ILD patients who underwent at least 2 pulmonary function tests (PFTs) with an interval of at least 24 months, as the screening period, between January 2009 and December 2015 were retrospectively reviewed. The deterioration of forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLco) was calculated based on PFT data and screening period. The definition of PF-ILD was1.relative decline of 10% or more in FVC per 24 months or2.relative decline in FVC of 5% or more with decline in DLco of 15% or more per 24 months.Medical records of 34 patients with idiopathic pulmonary fibrosis (IPF), 11 patients with non-IPF, PF-ILD, and 46 patients with non-IPF, non-PF-ILD were retrospectively analyzed. Patient characteristics, pharmacologic or non-pharmacologic treatment status, and prognosis were compared between the IPF and non-IPF groups and between the non-IPF, PF-ILD and non-IPF, non-PF-ILD groups.Eleven patients (19.3%) showed a progressive phenotype in the non-IPF group. The pulmonary function data at the first PFT were worse in non-IPF, PF-ILD patients than in non-IPF, non-PF-ILD patients. There were no differences in the proportion of patients who were observed without pharmacologic treatment or of those receiving pharmacologic treatment between the non-IPF, PF-ILD and non-IPF, non-PF-ILD groups. Low %FVC at the first PFT and the usual interstitial pneumonia-like fibrotic pattern on high-resolution computed tomography were risk factors for PF-ILD in the non-IPF group. The mortality in the non-IPF, PF-ILD group was significantly worse than that of the non-IPF, non-PF-ILD group and was as poor as that of the IPF group. Multivariate logistic analysis showed that aging and low %DLco at the first PFT were risk factors for mortality within the non-IPF group.The prognosis of non-IPF, PF-ILD patients was as poor as that of IPF patients. Non-IPF, PF-ILD patients require more intensive treatment before disease progression. Recently, these fibrosing ILDs were grouped under the term “progressive fibrosing ILDs” (PF-ILDs).Idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia (IIPs), is a chronic and progressive fibrosing interstitial lung disease (ILD) of unknown etiology. In most clinical trials or observational studies, the rate of decline in forced vital capacity (FVC) was used to assess disease progression in ILD patients.–16 Furthermore, gas exchange parameters, including the diffusing capacity of the lung for carbon monoxide (DLco), worsening of symptoms, exercise capacity, deterioration in health-related quality of life, the extent of lung fibrosis on high-resolution computed tomography (HRCT), or the initiation of supplemental oxygen therapy, were used to assess disease progression.The definitions of disease progression of ILD vary across studies. non-IPF, PF-ILD was defined as a relative decline in FVC, worsening of symptoms, or increased extent of fibrotic changes on chest HRCT within 24 months. Nintedanib demonstrated slowing disease progression in non-IPF, PF-ILD in INBUILD trials, and now, nintedanib is one of the essential treatment options in ILDs with a progressive phenotype. However, the clinical features of non-IPF, PF-ILD in a real-world setting remain unknown.In INBUILD trials,In this study, we retrospectively analyzed the clinical features of non-IPF, PF-ILD patients based on the deterioration of their pulmonary function in the past 24 months in a real-world setting.22.1This study was approved by the Ethics Committee of the Shinshu University School of Medicine  and was performed in accordance with the Declaration of Helsinki and its subsequent amendments. The need for obtaining patient written informed consent was waived, owing to the retrospective nature of this study.This retrospective, single-center study reviewed the medical records of ILD patients, including those with IIPs, CTD-ILDs, and other related ILDs, such as drug-induced lung disease, who underwent at least 2 pulmonary function tests (PFTs) with an interval of at least 24 months, as the screening period, at the Shinshu University Hospital between January 2009 and December 2015. For patients who underwent multiple PFTs during the same period, the PFT performed closest to January 2009 was defined as the first PFT, and that performed at 24 months after the first PFT was defined as the second  were diagnosed as having ANCA-associated ILDs.,24 Patients with sarcoidosis and drug-induced pneumonitis were diagnosed according to the respective criteria.,26 In this study, PF-ILD was defined only by the deterioration of pulmonary function, in consideration of data loss such as subjective symptoms or deterioration of fibrosis on HRCT. In particular, PF-ILD was defined as theThe clinical, radiological, and pathological findings and disease behaviors were reviewed, and ILD diagnoses were made in a multidisciplinary discussion. IPF was diagnosed according to the 2018 Clinical Practice Guideline.1.relative decline of 10% or more in FVC per 24 months and2.the relative decline in FVC of 5% or more with decline in DLco of 15% or more per 24 months, with reference to recently suggested definitions.A flow diagram showing patient selection is presented in Figure 2.2 When the HRCT findings met criteria A, B, and C as described below, or criteria A and C or B and C, the HRCT pattern was defined as a UIP-like fibrotic pattern. These HRCT criteria were as follows: A) definite honeycomb lung destruction with basal and peripheral predominance; B) presence of reticular abnormality and traction bronchiectasis consistent with fibrosis, with basal and peripheral predominance; and C) absence of atypical features, specifically nodules and consolidation. Ground glass opacity, if present, was less extensive than the reticular opacity pattern.Two radiologists (S.K. and M.M.) independently reviewed the chest HRCT findings during the first PFT and determined whether the patient showed a usual interstitial pneumonia (UIP)-like fibrotic pattern or a non-UIP pattern without referring to clinical information. In case of disagreement on HRCT findings between the 2 reviewers, they discussed and resolved the case by mutual consensus. The UIP-like fibrotic pattern was defined according to the radiological criteria in the INPULSIS trials.2.3,29 The composite physiologic index (CPI),,31 representing a combination of pulmonary ventilation and diffusing capacity, was also calculated based on the pulmonary function data.In this study, patients underwent PFTs, including spirometry and measurements of the DLco, functional residual capacity, total lung capacity (TLC), and residual volume, using a pulmonary function testing system , as previously reported.Relative decline in FVC and DLco per 24 months was calculated based on PFT data and screening period. For example, when the percent predicted FVC (%FVC) at the first PFT was 80% and that at the second PFT 30 months later was 72%, the relative decline in FVC per 30 months was calculated as 10%, and the relative decline in FVC per 24 months was calculated as 8% . Statistical significance was established at P values of <.05.Continuous data are presented as median and range, and categorical data are presented as the number in each group. While the Mann–Whitney 33.1P = .04). The respective pulmonary function data and CPI at the first PFT in the IPF and non-IPF groups did not differ significantly, except for percent predicted TLC (%TLC). The %TLC and %DLco at the second PFT were poorer in the IPF group than in the non-IPF group . CPI at the second PFT was also poorer in the IPF group than in the non-IPF group (P < .01). Relative decline in FVC ≥10% per 24 months was observed in 6 (17.6%) patients in the IPF group and 9 (15.8%) patients in the non-IPF group. Relative decline in DLco ≥15% per 24 months was observed in 17 (50.0%) patients in the IPF group and 11 (19.3%) patients in the non-IPF group.The clinical characteristics of the IPF and non-IPF patients are described in Table 3.2P = .03). In the non-IPF group, 2 of the 7 patients (28.6%) with idiopathic non-specific interstitial pneumonia (iNSIP), 2 of the 16 patients (12.5%) with unclassifiable IIPs, and 3 of the 22 patients (13.3%) with CTD-ILD were diagnosed with non-IPF, PF-ILD. The %FVC and %TLC at the first PFT in the non-IPF, PF-ILD group were poorer than those of the non-IPF, non-PF-ILD group. Additionally, pulmonary function data and CPI at the second PFT in the non-IPF, PF-ILD group were also poorer than those of the non-IPF, non-PF-ILD group.Patients with non-IPF were further divided into the non-IPF, PF-ILD and non-IPF, non-PF-ILD groups. Eleven of 57 (19.3%) patients with non-IPF showed a progressive phenotype (9 patients showed the relative decline of 10% or more and 2 patients showed the relative decline in FVC of 5% or more with decline in DLco of 15% or more per 24 months). Clinical characteristics of the non-IPF, PF-ILD and non-IPF, non-PF-ILD patients are described in Table P = .02 and P = .03, respectively). However, in the multivariate analysis, these were not significant risk factors for PF-ILD in the non-IPF group.We examined the risk factors for PF-ILD in 57 non-IPF patients Table . The log3.3Treatment status throughout the study period for each disease of the IPF and non-IPF groups are shown in Table 3.4P = .02). Similarly, the non-IPF, PF-ILD group had a significantly worse survival than the non-IPF, non-PF-ILD group (P = .01). A similar trend was demonstrated in the survival analysis between the IPF and non-IPF, PF-ILD groups.The Kaplan–Meier curves of overall survival in each disease group from the first PFT are shown in Figure P < .01, P = .02, and P = .03, respectively). In a multivariate analysis, aging and low %DLco at the first PFT were independent risk factors for mortality .We identified the risk factors for mortality in 57 non-IPF patients by performing a Cox proportional hazards regression analysis Table . The uni4This study showed 3 main findings regarding the clinical characteristics of patients with non-IPF, PF-ILD. First, the pulmonary function data at the first PFT were worse in the non-IPF, PF-ILD than in the non-IPF, non-PF-ILD group. Second, approximately half of patients with non-IPF, PF-ILD underwent observation without pharmacologic treatment, despite the deterioration of their pulmonary function. Last, the prognosis of the non-IPF, PF-ILD group was significantly worse than that of the non-IPF, non-PF-ILD group and was as poor as that of the IPF group. unclassifiable ILD, CTD-ILDs, including rheumatoid arthritis-associated ILD, systemic sclerosis-associated ILD, chronic hypersensitivity pneumonitis, and exposure-related disease.,13 The definition of PF-ILD varies across studies; therefore, there is no consensus on the definition of disease progression in patients with ILDs. Nevertheless, FVC decline has been used in some trials for IPF patients,,36 and is a well-established predictor of mortality. A recent phase III study, the INBUILD trial, demonstrated slower disease progression in non-IPF, PF-ILD patients with nintedanib treatment. This trial recruited non-IPF patients showing diffuse fibrosing lung disease with >10% extent of fibrosis on HRCT scans who met the criteria for disease progression at 24 months before screening, based on a relative decline of 10% or more in FVC, relative decline in FVC of 5% or more along with the increased extent of fibrosis on HRCT, relative decline in FVC of 5% or more along with worsening of respiratory symptoms or worsened respiratory symptoms and increased extent of fibrosis on HRCT. Recently, a definition of PF-ILD was proposed by adding the criteria of relative decline in FVC of 5% or more with a decline in DLco of 15% or more per 24 months to the INBUILD criteria. In this study, we defined PF-ILD according to the deterioration of the pulmonary function in the past 24 months.The term “PF-ILD” describes chronic fibrosing ILDs and mostly includes IPF, iNSIP, with varied diagnostic concordance across multidisciplinary teams. In this study, chronic hypersensitivity pneumonitis might have been diagnosed as IPF or unclassifiable IIPs in our multidisciplinary discussion.In our study, 11 of 57 patients with non-IPF were diagnosed with ILD with a progressive phenotype (PF-ILD). Here, PF-ILDs included iNSIP, unclassifiable IIPs, pleuroparenchymal fibroelastosis, CTD-ILDs, and ANCA-related ILDs. Chronic hypersensitivity pneumonitis was not identified in this study; however, some cases of chronic hypersensitivity pneumonitis show a progressive phenotype. The diagnosis of chronic hypersensitivity pneumonitis is challenging, meaning, these patients can now be treated with antifibrotic agents. To avoid FVC decline, early treatment is essential.First, in the present study, the pulmonary function at the first PFT was worse in the non-IPF, PF-ILD group than in the non-IPF, non-PF-ILD group. Low %FVC may be a risk factor for PF-ILD in the non-IPF group in addition to the UIP-like fibrotic pattern on HRCT. Thus, the pulmonologist should administer additional treatment before disease progression. Nintedanib has been shown to slow the FVC decline in non-IPF, PF-ILD patients;Second, 6 of 11 patients (54.5%) in the non-IPF, PF-ILD group were observed without pharmacologic treatment, despite showing the deterioration of their pulmonary function. In these cases, there is a possibility that pharmacologic treatment was not administered due to concerns for adverse events of immunomodulation treatment. In addition, these patients may have only been observed because there was no effective treatment option so far. Nintedanib might be expected to be effective in these cases.,6 which could be due to the difference in the inclusion criteria. The eligible patients in this study underwent PFT including DLco measurements twice or more for at least 24 months apart. Since patients with low FVC cannot undergo DLco assessments, ILD patients with rapid disease progression were not eligible for this study. Although the criteria were similar in the non-IPF group, IPF patients with rapid disease progression, that is, a poor prognosis, were not included in this study.Third, the prognosis of patients with non-IPF, PF-ILD was as poor as that of IPF patients. Aging, low %DLco, and PF-ILD were risk factors for mortality in the non-IPF group. The prognosis of IPF patients in this study was better than that previously reported, They reported that 168 of 617 (27.2%) patients with non-IPF, ILD had progressive fibrosing phenotypes. This result was higher than our study; however, the definition of progressive phenotypes used in their study was based on INBUILD trial. Differences in the definition of progressive phenotypes might therefore have contributed to the difference in the results. Third, this was a retrospective study; thus, whether the management and assessment for disease behavior were proper or not was uncertain. A prospective study design with well-defined endpoints and a larger sample size is therefore advocated. Despite these limitations, we demonstrated the clinical characteristics of non-IPF, PF-ILD patients and showed that their prognosis was as poor as that of IPF patients.Our study had some limitations. First, several patients with rapid disease progression who could not undergo follow-up PFTs were not included in this study; therefore, their prognosis might have been better than the actual prognosis of the IPF and non-IPF, PF-ILD groups. Second, this was a single-center study with a small sample size. Recently, Nasser et al reported the clinical characteristics of patients with PF-ILD other than IPF in a real-world setting.In conclusion, the prognosis of patients with non-IPF, PF-ILD was as poor as that of IPF patients. Low %FVC may be a risk factor for PF-ILD in the non-IPF group. Non-IPF, PF-ILD patients require more intensive treatment before disease progression, and nintedanib might be an option.www.editage.jp) for English language editing.We would like to thank Editage (Conceptualization: Hiroshi Yamamoto, Masayuki Hanaoka.Data curation: Masamichi Komatsu, Hiroshi Yamamoto, Yoshiaki Kitaguchi, Satoshi Kawakami, Mina Matsushita, Takeshi Uehara, Yosuke Wada, Takumi Kinjo, Takashi Ichiyama, Kazuhisa Urushihata, Atsuhito Ushiki, Masanori Yasuo.Formal analysis: Masamichi Komatsu.Funding acquisition: Masamichi Komatsu.Investigation: Masamichi Komatsu.Methodology: Masamichi Komatsu.Project administration: Hiroshi Yamamoto.Supervision: Hiroshi Yamamoto, Masayuki Hanaoka.Validation: Masamichi Komatsu.Visualization: Masamichi Komatsu.Writing – original draft: Masamichi Komatsu.Writing – review & editing: Hiroshi Yamamoto, Masayuki Hanaoka.


This experimental study investigates the mechanical properties of polymer matrix composites containing nanofiller developed by fused deposition modelling (FDM). A novel polymer nanocomposite was developed by amalgamating polycarbonate-acrylonitrile butadiene styrene (PC-ABS) by blending with graphene nanoparticles in the following proportions: 0.2, 0.4, 0.6, and 0.8 wt %. The composite filaments were developed using a twin-screw extrusion method. The mechanical properties such as tensile strength, low-velocity impact strength, and surface roughness of pure PC-ABS and PC-ABS + graphene were compared. It was observed that with the addition of graphene, tensile strength and impact strength improved, and a reduction in surface roughness was observed along the build direction. These properties were analyzed to understand the dispersion of graphene in the PC-ABS matrix and its effects on the parameters of the study. With the 0.8 wt % addition of graphene to PC-ABS, the tensile strength increased by 57%, and the impact resistance increased by 87%. A reduction in surface roughness was noted for every incremental addition of graphene to PC-ABS. The highest decrement was seen for the 0.8 wt % addition of graphene reinforcement that amounted to 40% compared to PC-ABS. Thermoplastics and thermoset plastics are two categories of polymer materials that are used based on application. Thermoplastics polymers are the most abundantly available materials that are used in the FDM process due to lower cost and lower melting temperature . The mosThree-dimensional printing, also known as additive manufacturing (AM), is gaining popularity among industrialists and researchers. According to Ford et al., fused deposition modeling (FDM), a significant shareholder of AM in the current market, uses polymer in the form of filament as a raw material to develop either prototype or functional models. Any complex model can be quickly created using FDM. The printed model is created in any CAD format and then converted to STL file format, which is then sliced and fed into the 3D printer. The option of using AM opens up a wide range of possibilities for improving design properties and printing speed . Keshava2 compared to pure ABS; however, tensile strength decreased for jute fiber and TPE [Melenka et al. conducted an experimental study with a polymer matrix as ABS and a reinforcement of Kevlar fibers. To test the tensile properties, the composite was created with different volume percents of 4.04 percent, 8.08 percent, and 10.1 percent. It was discovered that increasing the filler content increased the Young’s modulus and tensile strength . Perez e and TPE . Vijay e and TPE .2 could improve thermal stability, and Weng Z. et al. [Brennan et al. investigated the improvement of mechanical properties for low loading of graphene oxide with carboxyl and hydroxyl functional groups. The addition of nanofiller material would significantly improve the required properties for FDM-printed parts. The improvement in the multifunctional property was observed for the addition of less than 0.1 wt percent of GO . Lin et . et al.  investigVijay T. et al. recently investigated the filament characterization of a PC-ABS filament reinforced with graphene nanofiller. The filler was added in increments of 0.2, 0.4, 0.6, and 0.8 weight percent. The SEM and elemental mapping provided useful information about the dispersion of filler content in filaments produced by the twin-screw extrusion process. The presence of filler had no effect on the diameter of the extrusion wire . The devCertain polymers have distinct properties and serve as a tough material. Polycarbonate (PC) is one such polymer with a high toughness and resistance value. It can behave as a brittle material under certain strain conditions. The addition of a limited amount of ABS to PC resulted in balancing certain properties and improvements in mechanical stability and economic value . PolymerThe extraction of PC-ABS in the form of filament, which is required for FDM, could be performed in a smooth flow while retaining the desired properties. To create the composite, graphene, a novel material with extremely high mechanical and thermal properties, is used as nanofiller. In the current study, graphene is used as a filler material in the PC-ABS matrix at 0.2, 0.4, 0.6, and 0.8 wt percent. Physical and mechanical properties of the developed polymer composite are tested.The following PC and ABS in the form of pellets were mixed in the ratio 70:30. The parts to be fabricated by polymers were obtained in the form of pellets. As shown in The addition of graphene has exhibited proper amalgamation with matrix material, leading to even surface diameter of the filament. The macroanalysis showed no cracks or flaws in the developed filament, and microanalysis did not exhibit any visible defects. The addition of graphene reinforcement to the matrix showed the diameter readings to be similar to that of pure PC-ABS without any effect on surface smoothness.JSM 7100F Jeol model field emission scanning electron microscope was used for microstructure analysis of graphene. Scanning electron microscope  was used for SEM and EDAX studies.Philips X’Pert Pro X-ray diffractometer was used for taking XRD patterns on developed composites. This ray is diffracted from the specimen and is recorded in the acquisition software.TM Raman system, Bangalore, India. First, a 785 nm wavelength laser with a power of 5–300 mW was implemented in excitation of the sample. A 100 micron laser spot size was noted. The system comprises TE cooled, efficient CCD detector arrays which are cooled at −20 °C. A vial holder is used to place the sample. High sensitivity, resolution, and stability of the machine are met with utilization of USP monograph 1120. Rayleigh scatters are filtered by deep blocking laser, which helps isolate Raman scatter for valuable molecular analysis. The Raman spectra were recorded with RSIQ software. The resolution of the spectra is ~6 cm−1. The accumulation time is 5 s.The Raman spectrum was recorded with Peak SeekerPro3 enclosed chamber was the build volume of the printer. Optimal parameters were chosen for the development of models as follows: infill density of 100%, layer thickness of 0.1 mm, shell thickness of 0.4 mm, top and bottom layer thickness of 1.2 mm, speed of 5 mm/s, the orientation of 45°, the temperature of the nozzle for PC-ABS fabrication maintained at 240 °C and varied for other proportions, and bed temperature maintained at 80 °C. The 3D printer used for printing is shown in FDM follows a process of developing the models by the addition of material layer-wise. FDM is one of the most widely used 3D manufacturing techniques used for the development of polymer parts. The 3D model is developed through a CAD file. Any complex part can be easily developed through this process . A pramaSurface roughness (Ra) was measured for the FDM parts using Miyu surface roughness tester (M35:2010), Bangalore, India. The specimen of 10 mm × 10 mm × 20 mm was developed for measurement of surface roughness Ra, through which three trials were conducted at various lengths of the surface as shown in ASTM D638 standard procedure was implemented for conduction of tensile test , make ofFDM parts’ ability to hold the load with the increase in filler content was analyzed using Izod impact test following a procedure of ASTM D4812 . The tes−1. The D band indicates the presence of disorder either in vacancies, grain boundaries, and carbon content’s amorphous nature. Johra et al. [The obtained XRD pattern for graphene is represented in a et al.  obtainedThe studies of the dispersion of nanofiller have stated that graphene dispersion occurs in the styrene-acryolnitrile phase and not in the polybutadiene phase of ABS . Gao et d spacing of 3.36 Ᾰ. Thus, the presence of peaks proves the existence of graphene in the PC-ABS matrix, with multilayer graphene having a specified d spacing. The FESEM image shown above in The first layer of filament that is laid on the heating bed from the nozzle forms a binding zone between the adjacent filament surfaces that are laid one above the other; gradually, the diffusion occurs because of variation in temperature between the adjacent layers and also between the layer and the bed, the rapid cooling of the layers leading in enhancing the bonding between the layers ,29. HoweThe roughness (Ra) average values are used to compare the roughness with different specimens and are plotted in the graph as shown in Compared to pure PC-ABS addition of 0.2 wt % of graphene, it has a decrement of 25.8% in surface roughness. Similarly, the decrement trend is followed with addition of reinforcement. The addition of 0.4 wt % graphene PC-ABS saw a reduction of 11.1% in comparison to 0.2 wt % graphene addition. Similarly, a reduction of 12.5% was noted for 0.6 wt % addition of graphene compared to 0.4 wt % addition. The highest decrement is seen for the 0.8 wt % addition of graphene reinforcement that amounts to 40% compared to PC-ABS. The calculation of error bars was performed by considering the standard deviation and dividing it by square root of the overall measurements that constitute the mean.ASTM D638 testing method was followed to determine the ultimate tensile strength of the fabricated part. The graph in The increased tensile strength with graphene addition is attributed to the massive intermolecular linkages and reinforcement alignment . This prSeyeon et al. developed a polymer nanocomposite using ABS as a matrix and iron and copper as filler material. The tensile strength analysis showed that the specimens printed with 3D printing technology would have better tensile properties with increased fill density. An impressive result was revealed such that as the increment in metal filler increases above a specific value, the decrement in tensile strength is observed .The impact strength of pure PC-ABS had a value of nearly 1 J, which increased with the incremental addition of graphene reinforcement; 0.4 wt % addition of graphene gave an incremental value of nearly 4 J, an increase of 75% with impact resistance. The highest value of 87% was observed for the addition of 0.8 wt % of graphene. Suarez et al. have stated that the addition of PC to ABS increased the developed specimens’ impact strength . An incrThe incorporation of graphene into the matrix tends to inhibit deformation and ductile mobility of polymer molecules while also absorbing energy during crack propagation. Fewer flaws in composites result from fewer void spaces, which improve composite impact strength. The following conclusion has been derived from the experimental study carried out in determining the mechanical properties of polymer nanocomposites developed by FDM:Fixed filament of required diameter was successfully carried out for PC-ABS + graphene filament by compounding and twin-screw extrusion.The PC-ABS and composite specimens have been tested perpendicular to printing, as this direction offers the highest mechanical properties in the 3D printing process.Microstructure studies indicate proper dispersion of graphene in the PC-ABS matrix and elemental mapping suggests no formation of aggregates.Reduction in surface roughness was noted for every incremental addition of graphene to PC-ABS. A 0.2 wt % addition of graphene showed a decrement of 25.8% in comparison to PC-ABS. The highest decrement was seen for the 0.8 wt % addition of graphene reinforcement that amounted to 40% compared to PC-ABS.The increment in tensile strength is in linear trend with the addition of graphene. With a maximum addition of 0.8 wt % graphene in this experiment, the tensile strength increased by 57% compared to pure PC-ABS. A 34.32% increase in yield strength has been noted for PC-ABS + 0.8 wt % when compared to pure PC-ABS. Percentage elongation was reduced with every increment of graphene added to the PC-ABS matrix.An increase in resistance to breaking energy with the addition of graphene has been observed.Graphene nanosheets addition has significantly improved the physical and mechanical properties of FDM parts.


Saccharomyces cerevisiae. Markedly, Fkh1 impinges chromatin architecture by recruiting large regulatory complexes. Implication of Fkh1 with transcriptional corepressor complexes remains largely unexplored. In this work we show that Fkh1 directly recruits corepressors Sin3 and Tup1 (but not Cyc8), providing evidence for its influence on epigenetic regulation. We also identified the specific domain of Fkh1 mediating Sin3 recruitment and substantiated that amino acids 51–125 of Fkh1 bind PAH2 of Sin3. Importantly, this part of Fkh1 overlaps with its Forkhead-associated domain (FHA). To analyse this domain in more detail, selected amino acids were replaced by alanine, revealing that hydrophobic amino acids L74 and I78 are important for Fkh1-Sin3 binding. In addition, we could prove Fkh1 recruitment to promoters of cell cycle genes CLB2 and SWI5. Notably, Sin3 is also recruited to these promoters but only in the presence of functional Fkh1. Our results disclose that recruitment of Sin3 to Fkh1 requires precisely positioned Fkh1/Sin3 binding sites which provide an extended view on the genetic control of cell cycle genes CLB2 and SWI5 and the mechanism of transcriptional repression by modulation of chromatin architecture at the G2/M transition.Transcription factors are inextricably linked with histone deacetylases leading to compact chromatin. The Forkhead transcription factor Fkh1 is mainly a negative transcriptional regulator which affects cell cycle control, silencing of mating-type cassettes and induction of pseudohyphal growth in the yeast The online version contains supplementary material available at 10.1007/s00294-021-01158-3. S. cerevisiae encodes four Fox proteins among which Fkh1 and Fkh2 (Forkhead homolog) are important regulators of the CLB2 cluster of cell cycle-regulated genes, responsible for the transition of the G2/M boundary (Jorgensen and Tyers ForkHead Associated (FHA) domain which is required for protein–protein-interactions with an emphasis on recognition of phosphopeptides . Complete genotypes of all used strains are available in the supplementary file, Table For ChIP analyses, derivatives of Escherichia coli expression plasmids  encoding various glutathione S-transferase (GST) fusions were constructed. Length variants of coding regions of the FKH1 gene were amplified by PCR and fused behind GST. Similarly, HA-tagged length variants of Sin3 representing PAH domains, Cyc8 and Tup1 were expressed in yeast using plasmid p426-MET25HA  has been described , pFK77  and pRAR110  derived from pASK-IBA5  were used. To confirm authenticity of gene fragments obtained by PCR, GST fusions which encode minimal length variants of Fkh1 were verified by DNA sequencing . Plasmid names and fused sequences are mentioned in legends of figures and are described in detail in supplementary file Table FKH1 gene (Δfkh1::LEU2). To construct this plasmid, flanking sequences upstream and downstream of the respective coding region were amplified by PCR and inserted on both sides of LEU2 selection marker, allowing total deletion of the FKH1 reading frame.To perform interaction assays, E. coli strain BL21 (Stratagene/Agilent). The tac promoter controlling GST fusion genes was induced with 1 mM IPTG. Similarly, tetR-dependent gene expression was activated by 0.2 mg/l anhydrotetracycline. Derepression of MET25-dependent gene fusions was achieved by cultivating yeast transformants in the absence of methionine. GST fusion proteins synthesized in E. coli were released by sonication, immobilized on glutathione (GSH) sepharose and subsequently incubated with yeast or bacterial total protein extracts containing HA fusions. To avoid unspecific interactions, protein extracts were pre-cleared by treatment with GSH Sepharose beads prior to incubation with GST fusions. After the release of GST fusions with free GSH (10 mM), eluates were separated by SDS-PAGE and proteins transferred to a filter. Following incubation with anti-HA-peroxidase conjugate, HA fusion proteins were detected with POD chemiluminescent substrate .GST- and HA-tagged proteins used for interaction assays by affinity chromatography were synthesized by MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal80Δ UASGAL2-ADE2 LYS2::UASGAL1-HIS3 met2::UASGAL7-lacZ; James et al. BDTRP1) while Fkh1 domains  were inserted in pGAD-C1 (2 µm GAL4ADLEU2). Double-transformed strains containing both types of fusion plasmids were first selected on a medium lacking leucine and tryptophan (-L-T) and subsequently transferred to a medium devoid of adenine (-L-T-A).To perform two-hybrid assays, strain PJ69-4A was used (fkh1 (L74A and I78A) and the absence of any other change in the plasmids obtained .To alter selected residues in the coding region of Fkh1, the QuikChange site-directed mutagenesis kit of Stratagene was used. To obtain mutations within the FHA domain, plasmid pRAR73 containing the Fkh1 coding region was used. To replace selected residues against alanine, we used pairs of mutagenic primers introducing a GCA codon instead of the natural codon, flanked by 15–19 nucleotides on both sides. DNA sequencing was used to confirm the presence of the desired mutant alleles of FKH1 was modified such that it expressed a His-tagged Fkh1 without alteration of gene copy number or control region. Tagging was performed by the transformation of strain C13-ABY.S86 with a gene-specific modification fragment and selection for resistance against geneticin. The modification fragment was amplified by PCR, using gene-specific primers and plasmid pU6H3HA as a template , FKH11 (SIN3-HIS6-HA3-kanMX) and its isogenic fkh1 derivative RAY5 (Δfkh1 SIN3-HIS6-HA3-kanMX) grew until mid-log phase and were treated with formaldehyde for 15 min.Chromatin immunoprecipitation (ChIP) analysis followed the procedure described by Cobb and Van Attikum . Chromosg to remove insoluble material and incubated for at least 4 h with His-Tag Dynabeads® . After elution of affinity-purified proteins and bound DNA with a buffer containing 300 mM imidazole, cross-linking was reversed by heating to 65 °C overnight. DNA was recovered and analyzed by PCR (29 amplification cycles), using specific primers for promoters of CLB2 (− 880/− 580), SWI5 (− 420/− 170) or ACT1 .The crosslinking reaction was subsequently quenched for 5 min by the addition of glycine to a final concentration of 125 mM. After lysis, cells were sonicated five times for 30 s to shear chromatin, using a Bandelin Sonoplus UW 70 microtip (35% power). After sonication, lysates were centrifuged for 10 min at 16,000 Based on the methods of homology, the secondary structure of Fkh1 protein was predicted using an accurate web-based predicting tool Phyre2 , selecting on SCD -Ura -Leu medium. Transformants were cultured in double-selective medium at 30 °C until mid-logarithmic growth phase and then specific β-galactosidase activities were determined in the crude extract. The empty repressor test plasmid pRT-lexA was used as a control.Effector plasmid encoding Fkh1 (full length) fused with the DNA binding domain of lexA (supplementary Table 1b) was transformed into two strains containing integrated reporter genes -Fkh1 fusion protein  was synthesized in E. coli and subsequently bound to glutathione (GSH) sepharose. A protein extract from S. cerevisiae transformants containing epitope-tagged Sin3 (full length) was added to this affinity matrix. After intensive washing with increasing stringency, specifically bound protein was eluted by the addition of free GSH. As is shown in Fig. 3-Sin3 could be bound by GST-Fkh1 under stringent conditions but not by GST. Thus, Fkh1 may execute its function by recruiting the general corepressor Sin3.A high-throughput characterization of yeast protein complexes by mass spectrometry provided evidence for a physical interaction of Fkh1 with corepressor Sin3  can bind to PAH1 and PAH2 of Sin3. To inspect the minimal core domain of Fkh1 in charge of Sin3 recruitment, we tested shorter Fkh1 length variants which revealed that aa 81–160 of Fkh1 are able to mediate Sin3 binding. More precise, 75 amino acids at the N-terminus  turned out as the domain which is sufficient to interact with PAH1 and PAH2 of Sin3.As is apparent from Fig. Vice versa, the physical map of interacting domains within Sin3  and PAH2 (aa 301–888) were fused with the DNA-binding domain (BD) of Gal4. Length variants of Fkh1 which in vitro showed Sin3 binding were fused with the Gal4 transcriptional activation domain (AD). In vivo Sin3-Fkh1 interactions should reconstitute a functional Gal4 activator being able to stimulate expression of the 51–125 minimal binding domain which has displayed in vitro interaction with PAH2 of Sin3 is able to show in vivo binding as well, proving the interaction between Fkh1 core domain (aa 51–125) and PAH2 of Sin3.As is apparent from the results summarized in Table In contrast to in vitro results, BD fusions of Sin3  in combination with AD fusions of Fkh1 (aa 81–160 and aa 51–125) were unable to mediate growth on medium lacking adenine. Presumably, formation of functional interaction domains in vivo is prevented with certain length variants due to failure of correct protein folding. In summary, use of the in vivo two-hybrid system confirmed that PAH2 domain of Sin3 exclusively interacts with Fkh1.Based on the methods of homology, the secondary structure of the full length of Fkh1 protein was predicted to consist of 15 β-sheets and 7 α-helix structures Fig. a. The FkAs aforementioned, the N-terminus of Fkh1 residues 51–125) with its FHA domain is able to bind PAH2 of Sin3 in vitro and in vivo. Within the minimal identified domain of Fkh1, a short pattern of alternating hydrophobic amino acids which extends from aa 67–80 could be identified. To scrutinize the significance of these residues for interaction with Sin3 (and consequently for regulated expression of Fkh1 target genes), we implemented a site-directed mutagenesis at selected positions leading to the replacement of large hydrophobic amino acids to alanine [single mutations L74A and I78A; pointed out with arrows  were completely defective for interaction with Sin3. In vivo binding of Fkh1 missense variants to Sin3 was again investigated by the two-hybrid system.The impact of alanine substitutions in Fkh1 domain aa 51–125) on binding to Sin3 was subsequently investigated in vitro (by GST pull-down) and in vivo (two-hybrid system). To assay for in vitro interaction of Fkh1 missense variants with Sin3, GST-Fkh1 fusions (comprising aa 51–125) with mutational variants L74A and I78A were synthesized in 1–125 on 51–125 fusion used above together with its mutational variants L74A and I78A were co-transformed into strain PJ69-4A, containing a plasmid encoding BD-Sin3301–888 (comprising PAH2). In contrast to the wild-type minimal binding domain (Fkh151–125), Fkh1 variants L74A and I78A were unable to activate the GAL2-ADE2 reporter gene, resulting in the failure of transformants to grow in the absence of adenine  was employed to monitor directly the occupancy of particular promoters by both regulatory proteins. A substantial body of data points to the significant role of Fkh1 in the regulation of the cell cycle  during the G2/M phase of the mitotic cell cycle .Fkh1 regulates the expression of the CLB2 and SWI5 are key players in cell cycle regulation  and then Sin3 recruitment to target genes was investigated again. As is apparent from Fig. ∆fkh1 mutant strain although Sin3 was detected in the presence of an intact copy of Fkh1. Thus, Sin3 corepressor can be efficiently recruited to gene promoters when the Fkh1 protein is present.A strain encoding an epitope-tagged variant of Sin3  has been previously described  together with the Fkh1 coding region was constructed. Plasmid pRAR28 (lexABD-FKH1) together with the empty lexA plasmid pRT-lexA (negative control) was transformed into two strains containing integrated reporter genes . As shown in Fig. Although we here definitely confirmed the direct interaction between Fkh1 and Sin3, the in vivo significance of this result still remains open. According to previous findings, the assumption that Fkh1 may act as a repressor is obvious . However, a corresponding experiment with GST-Fkh1 and E. coli extract containing HA-tagged Tup1 revealed a direct interaction between Fkh1 and Tup1 , indicating that the repressor function of Fkh1 may be redundantly executed by two separate corepressor complexes.Previous studies have shown that repressor proteins may utilize more than a single corepressor complex to negatively affect transcription  was initially identified as a surrogate for SIR1 (silent information regulator one) gene as it recovered mating ability in a sir1 mutant . There was an initial study that highlighted Sin3 as an associated protein to Fkh1 through high-throughput mass spectrometric protein complex identification  and Ndd1 is dephosphorylated, Fkh proteins may instead recruit Sin3 which allows promoter access for various HDACs being associated with Sin3  domain. While the DNA binding domain of Fkh1 turned out as inessential for Sin3 corepressor recruitment, we could demonstrate that aa 51–125 precisely covering its FHA domain are sufficient to bind PAH1 and PAH2 of Sin3 in vitro. Moreover, in vivo experiments indicated the inability of Fkh1 aa 81–160 and aa 51–125 length variants to bind PAH1 domain of Sin3 which suggests that PAH2 could be the genuine docking site playing role in stabilization of Fkh1/Sin3 interaction. Importantly, the FHA domain (at least of Fkh2) has been also described as a binding site for phosphorylated Ndd1 (dependent on protein kinases Cdk-Clb and Cdc5) which is indispensable for transcriptional activation of Op-CYC1-lacZ reporter gene 4.6 fold in asynchronously growing cells. Similarly, Cti6 and Ume6 were able to repress transcription by factors of 5.6 and 7.0, respectively , indicating that Sin3 recruitment is Fkh1-dependent. Previously, Veis et al. . Future studies should investigate whether Fkh1 (and/or Fkh2) directly interact with Isw1 and Sir2 under both repression and derepression conditions. As a conclusion, the findings of previous work and the results described here are summarized by the scenarios depicted in Fig. To strengthen the in vivo significance of our results we also investigated chromatin occupancy of Fkh1 and Sin3 by ChIP. Although asynchronously growing cells were used, an association of Fkh1 and Sin3 with cell cycle-regulated promoters s et al.  reportedf et al.  could inSupplementary file1 (DOCX 5485 KB)Below is the link to the electronic supplementary material.


This review highlights the ocular applications of GFNsand systematically covers the most recent advances of GFNs’physicochemical properties, in vitro and in vivo ocular toxicity, and the possible toxicity mechanismsas well as provides some perspectives on the potential risks of GFNsin material development and biomedical applications.Graphene family nanomaterials(GFNs) are rapidly emerging for ocularapplications due to their outstanding physicochemical properties.Since the eyes are very sensitive organs and the contact between theeyes and GFNs in eye drops, contact lenses, intraocular drug deliverysystems and biosensors and even the workers handling these nanomaterialsis inevitable, it is necessary to investigate their ocular toxicitiesand physiological interactions with cells as well as their toxicitymechanisms. The toxicity of GFNs can be extremely affected by theirphysicochemical properties, including composition, size, surface chemistry,and oxidation level as well as dose and the time of exposure. Up tonow, there are several studies on the  The personnel who areworking with nanomaterials are at a high risk of exposure while theyare preparing or handling nanomaterials. Therefore, due to continuouslyexpanding demands of nanomaterials and increasing exposure to them,it is essential to evaluate the potential risks and hazards of nanomaterialsfrom the human health and safety perspective.In the field of pharmaceutical and biotechnology sciences, nanotechnologyis in high demand these days, leading to the desired products or therapeuticoutcomes. Development of nanomaterials has increasingly assigned alarge number of studies in academic and industrial groups to generateengineered advanced materials and biomedical systems.3 Designand development of novel therapy techniques using nanomaterials mightlead to novel therapeutic methods in ophthalmology. Among varioushuman organs, investigation of nanomaterials’toxicity on the eyes is crucially important due to high levels ofeye exposure with nanomaterials during manufacturing, use, and disposal.Moreover, recently, there are several outstanding reports on the applicationsof nanomaterials in ocular applications such as ocular drug delivery,eye drops, and contact lenses.6Among different nanomaterials, the carbonaceous materials, suchas graphene, carbon nanotube (CNT), and fullerene 2 are at 6 Graphene has received tremendous interest from academiato industries owing to its outstanding physicochemical and structuralproperties.11 The 2D sheet of carbon atoms in a honeycombed network provides graphenewith a large surface to volume ratio and high mechanical, thermal,and electrical properties.14 Generally, graphene, a singlesheet of graphite that is held together by a backbone of overlappingsp2 hybrid bonds, is a part of graphene family nanomaterials(GFNs). Graphene, few-layer graphene , graphite,reduced graphene oxide (rGO), graphene oxide (GO), graphene nanoplatelets(GNP), and graphene quantum dots (GQDs) are the most important GFNanalogs  generationin the A549 cell line.28 Also, the hemolyticpotential evaluation of different sizes of GO and rGO sheets showedthat the smaller one had a higher activity, while the minor hemolyticpotential was seen for the aggregated rGO.29 Morover, the size of GFNs is an important determining factor forsubcellular penetration. For instance, rGO nanoplatelets with averagelateral dimension of 11 nm can enter the nucleus of human mesenchymalstem cells (hMSCs) and induce more genotoxicity compared to 3 μmnanoplatelets. The micron-sized rGO nanoplatelets showed a high toxicityat high concentration (100 μg mL–1) after1 h exposure time, while the 11 nm rGO nanoplatelets translocatedto the nucleus and induced genotoxicity at a very low concentration(0.1–1 μg mL–1).30From a toxicological perspective,one of the prime physicochemical properties that influences GFNs’stoxicity is particle size. Decrease in size to a nano level leadsto more cellular uptake due to increasing the surface area and providingfurther sites for cellular interaction. Several papers reported thatthe mechanism and the efficiency of cellular uptake, the circulation,distribution, clearance, and toxicity of GFNs depend on the nanoparticles’size.2.232 The cell membranes’ performance and structure can be disruptedby GFNs due to their different surface chemistries. In addition, theyare able to stimulate receptors and activate mitochondrial pathwaysand induce apoptosis.35GFNs possess varioussurface chemistries that lead to different biological activities.The extent of oxidation (O/C ratio) imparts hydrophobic, partiallyhydrophobic, and intermediate hydrophilic surface chemistry for pG,GO, and rGO, which alter their dispersibility in different solventsand physiologic medium. The hydrophobicity of GFNs causes aggregationbecause of the π–π stacking between the layers.32Compared with rGO, GO is enriched by carboxylicacid and hydroxyl and epoxy functionalities at its edges and basalplane. Therefore, owing to the difference in their structures, GOhas more dispersion ability, binding sites, and higher activity, whichleads to differences in their toxicities. In a study, the ocular toxicityof GO and rGO was evaluated by exposure of GO and rGO to the conjunctivalsacs of mice. GO exposure led to observing intraocular inflammation,an incrassated corneal stromal layer, higher corneal stromal cellcounts, and TUNEL-positive cells in the cornea as well as iris neovascularization.Unlike GO, by rGO exposure, no specific ocular toxicity was observed,which can be due to their structure and physical characteristic differences.–1 for 2 h. The results showed a dose- and oxygen-dependentcytotoxicity response with greater cytotoxicity for pG. The toxicitylevels were correlated with an increase in functionalized oxygen content.The ROS measurements showed that the oxidized graphenes, low oxidizedgraphene (LOG), higher oxidized graphene (HOG), and GO generate higherROS levels comparing with pG. HOG produced higher superoxide molecules,but free radicals might be reduced due to cellular antioxidant mechanismsof graphene within the cells and its redox ability, preventing theobservation of high cytotoxicity.36Additionally, to compare the effect ofoxygen level of graphene-basedmaterial on its biocompatibility and cytotoxic potential, pG, GO,and low and high oxygen functionalized graphene containing 6.6% and24% oxygen contents were investigated. ROS generation evaluation wasexplored using PC12 cells at very low doses of 0.5, 1.0, and 5 μgmL37 Hence, negatively charged GO showed negligible internalization tononphagocytes;38 however, other reportshave proposed that the negatively charged nanoparticles internalizationinto nonphagocytic cells can occur when they bind to the cell surfacecationic sites and then they can be taken up by scavenger receptors.40 Due to the surface charge of nanomaterials, they can absorb severalproteins and form “coronas” with proteins in biologicalsystems. Certainly, the affinity and the mode of interaction of theproteins play a determining factor in the formation of protein corona.41 Negative charged GFNs cause more electrostaticinteraction with the proteins that can alter their circulation, distribution,clearance, and toxicity. Bovine serum albumin-coated GO relieved thecytotoxicity through decreasing its penetration to the cell membrane.Protein corona can decrease GO cellular uptake according to the reportedcell viability and cellular uptake results, leading to GO potentialcytotoxicity reduction.42Another feature that indicates the effect of surface chemistryon cellular uptake is surface charge, the key factor to induce toxicity.The overall charge of cell membrane is negative, which leads to easilybinding and ingestion of positive charge nanomaterials to the cellmembrane using electrostatic interaction.2 (the sample with higher oxidation level) exhibiteda higher cytotoxicity compared with the samples with lower oxidationlevels.43The oculartoxicity investigation of PEGylated GO (polyethyleneglycol (PEG)-GO) with various oxidation levels and/or surface chargeswas performed. The results revealed that while the surface chargecould change the aggregation status of GFNs, it did not affect thecytotoxicity of PEG-GO samples alone, and the oxidation level hada critical effect on the GO toxicity. Among different PEG-GO samples,GO-h-PEG-NH2.345 However, GO still aggregates in physiological buffers because ofthe charge screening effect of salts.46 Consequently, surface modification of graphene and GO is necessary,especially for biomedical applications.47 To improve the deficiencies in the structure of graphene and GO,scientists usually introduce active functional groups to improve theirdispersion in aqueous and biological media.50To controlthe behaviors of GFNs in biological systems and improve their biocompatibility,their surface chemistry plays an important role. Graphene is an extremelyhydrophobic compound since it does not have any oxygen-containinghydrophilic functionalities and has the π–π stackinginteractions that has crucial implications for its dispersion in water.GO is the most widespread of GFNs because it has been employed forthe synthesis of graphene-based nanomaterials on a large scale. GOshows good water dispersion stability and biocompatibility owing tothe presence of oxygen-containing functionalities on its edges andsurface, which make it a potential nanomaterial for various applications.51 Covalent and noncovalent modifications are the most extensivemethods for graphene and GO functionalization, aiming to improve theirbiocompatibility, physiological and colloidal stability, decreasetheir nonspecific binding to biological molecules and cells, and increasetheir in vivo pharmacokinetics ,3 cyclodextrin,18 poly(ε-caprolactone),44 poly,45 pluronic,59 amine,60 carboxyl,and dextran61 groups significantly decreasedtheir toxicity and improved their biocompatibility.Depending on different application purposes,various surface functionalization strategies have been used to improveGFNs biocompatibility to be used in biomedicine.kinetics 1.52−57 U3, and GO-PEG-NH2) were fabricated, and their toxicitieson ocular tissue were investigated. Among different PEGylated GO,the GO-PEG-NH2 showed the most toxicity to ocular cells.The obtained results of this study can be used for biomedical applicationsof GFNs in the future by decreasing their toxicity with the aim ofsuitable surface modifications.43 In anotherstudy, hydroxylated graphene (G-OH) was prepared, and its ocular biocompatibilitywas investigated and compare with GO. G-OH displayed some featuresof GO such as water solubility and processability, whereas it showedhigher electroactivity and biocompatibility with human retinal pigmentepithelium (RPE) cells than GO.62Toinvestigate the role of GFN functionalities in ocular toxicity,in a study, three kinds of PEGylated GO  and graphene foam (G-foam), forthe synthetic keratoprosthesis skirt and assessed their biocompatibilitiesby in vitro cell culture using human corneal stromalcells and an in vivo rabbit implantation model. For in vitro assessment, human corneal stromal fibroblasts werecultured on the surface of G-film, G-foam and pristine titanium (Ti)discs as a standard, and a tissue culture plastic surface (TCPS).Good biocompatibility with human stromal fibroblasts was observedfor G-film in terms of cell adhesion, viability, and proliferation.The number of cells was higher on G-films compared with TCPS control,and 10% more cell proliferation was seen on graphene in comparisonwith on Ti. Moreover, compared with Ti and G-film, the culture mediumcollected from fibroblasts seeded on G-foam demonstrated lower cytokines(IL-6 and IL-8), which can be due to a lower expression of cytokinesby cells, or adsorption of the inflammatory signal molecules withgraphene materials. No sign of infection, neovascularization, or inflammationwas seen by the implantation of G-film into rabbit stroma, confirmingshort-term biocompatibility of graphene with corneal cells and tissue,which can be developed for cornea tissue engineering.Until now, the toxicity of GFNs on some anteriorsegment of the eyes, including conjunctiva, cornea, iris, and lens,has been investigated. In patients with end-stage corneal blindness,synthetic keratoprostheses are required for visual rehabilitation.To this aim, Tan et al.87 The acute eye irritation tests were performed in this studyin albino rabbits based on the OECD guidelines. They investigatedthe influence of GO exposure to the ocular surface in vitro and in vivo, considering different concentrations(12.5–100 μg mL–1) and times of exposure(–1 of GO (–1) in Sprague–Dawley rats that was alleviatedby glutathione (GSH) (Organisationfor Economic Co-operation and Development (OECD) guidelines for theTesting of Chemicals (Test No. 405) can be used as a standard forinvestigation of acute eye irritation/corrosion. According to theseguidelines, eye irritation is defined as “... the productionof changes in the eye following application of a test substance tothe anterior surface of the eye, which are fully reversible within21 days of application”. Moreover, the eye corrosion is alsodefined as “... the production of tissue damage in the eye,or serious physical decay of vision, following application of a testsubstance to the anterior surface of the eye, which is not fully reversiblewithin 21 days of application”. Aiming to this guideline, thetime- and dose-dependent cytotoxicity of GO through oxidative stresswas reported by Wu et al. using human corneal epithelium cells (hCorECs)and human conjunctiva epithelium cells (hConECs).exposure12. Althoexposure6A. The o–1 of GO 6B. Howevne (GSH) 6C. There120In vivo and in vitro morphological and molecular biologicalanalysis revealed no significant ocular toxicity for rGO, while differentsigns of toxicity, such as intraocular inflammation, an incrassatedcorneal stromal layer, cell apoptosis in the cornea, iris neovascularization,and significant cytotoxicity of rat corneal epithelial cells wereseen by short-term GO exposure. After short-term GO contact with eyes,many lymphoid cells were produced, as shown by the HE staining results.Interleukin expression was induced by lymphoid cell proliferation.In addition, compared with the control group, in the GO treatmentgroup, the level of IL-6 and IL-8 expressions were considerably higher.In addition, the TNF-α expression level, which is one of theimportant inflammatory factors, was also evaluated in vivo using GO and rGO. In the GO model mice, the TNF-α contentwas considerably enhanced in the eye, while it did not change in therGO model mice. Therefore, the inflammatory response had been triggeredby increasing the IL-6, IL-8, and TNF-α in the GO model mice.Moreover, the oxygenation levels of eyes in the three groups of micewere indicated by the malondialdehyde (MDA) content. In addition,the in vitro results demonstrated that the main patternsof GO cytotoxicity during cell injury were necrocytosis, cell death,cell cycle disorder, and cell viability loss.In another study on GFNs, theright conjunctival sacs of the Kunmingmice  were exposed once per day for a total of 7 days to therGO and GO suspensions.2 sample, which had a higher oxidation level, exhibited highertoxicity. To understand the potential toxicity mechanisms and complicatedinteractions between GFNs and biological systems on a whole cell level,a study of gene expression profiles can be used as an important approach.Therefore, in this study, the gene expression profile was studiedand the results demonstrated that the accumulation of ROS inducedby GO-h-PEG-NH2 treatment was attributed to NDUFB9-mediatedbiological pathway.43The toxicity investigationof PEG-GO with various oxidation levelsand/or surface charges including positive, negative, and neutral chargeon hCorECs and intraocular cells (hRCECs) was performed. The resultsrevealed that the viability of both cell types decreased by increasingPEG-GO concentration. Among different PEG-GO samples, the GO-h-PEG-NH3.388 RPE cells exhibited >60% cell viability in GO solutionsand <8% LDH release. Although LDH release into the culture mediumupon cells showed a very low impact on cell morphology, after a long-timeculturing, the change was noticeable as well as aggregation of GO.Moreover, the results showed a negligible cell apoptosis (∼1.5%)with the addition of 100 μg mL–1 GO (similarto the control cells), indicating GO biocompatibility. The biocompatibilityof GO was also investigated in vivo by intravitreallyinjection of 0.1, 0.2, and 0.3 mg of GO into white rabbits’eyes. One eye was injected with GO, and the salt solution  was injected to the other eye of the same rabbit asthe control. As seen in The toxicity of GFNs on some posterior segment of theeye was also investigated, such as retina, macula, and optic nerve.The effect of GO on RPE cells in terms of the cell morphology, viability,membrane integrity, and apoptosis was examined by Yan et al., usingseveral techniques, such as optical micrography, cell counting kit-8,lactate dehydrogenase (LDH), and apoptosis assays.in vitro and the in vivo results obtainedin this study, it can be concluded that there is no severe GO toxicityon the eyes.88 However, still more studies,such as controlling GO aggregation by surface functionalization andGO genotoxicity as well as the impact of physicochemical propertieson the GO toxicity, should be considered before making a certain conclusionabout the safety of GO.In addition, the histopathology studies of the rabbits’eyes revealed that while a very low content of GO remained in theGO-injected eye, by comparing with the control eye, no retinal abnormalitywas seen. These observations suggested that by GO injection, no apparentdamage occurred to the retinal morphology. Considering both the 89 Hydroxylated graphene (G-OH) showed genotoxicity at 4100mg mL–1 concentration. Although the genotoxicityof G-OH in rabbits was decreased gradually through intravitreal injectionafter 4 weeks, it did not show damage to cell morphology, structure,and most parts of the eyes. It led to IOP, ERG, and retinal structurechanges as eyesight-related functions. The cytotoxicity mechanismresults also revealed that through endocytosis and exocytosis, G-OHcould penetrate into and out of the cytoplasm without any cell membranedamage.90In another research work, GO showedmitochondria damage and induceddevelopmental malformation of the zebrafish eyes.121 developedrGO membrane (rGOM) for ocular regenerative medicine application.For ocular tissue engineering, the used membrane should have cellularbiocompatibility and promote wound healing as well as show antimicrobialproperties. Because of these needed criteria, the in vitro and in vivo biocompatibility and genotoxicity ofrGOM were investigated using various human ocular cells. The resultsdemonstrated that no sign of cytotoxicity or genotoxicity was seenafter short-term exposure of rGOM compared with control group culturesand allowed the growth of different ocular cells. Although the obtainedresults of this study were very promising, a long-term follow-up periodand additional in vivo research are needed to findout whether rGOM can be a good candidate for treatment of ocular diseasesor not.GFNs can be used in ophthalmologyto treat different ocular disorders.Recently, Zambrano-Andazol et al.44.1123 Ocular drug delivery using GFN-containing systems has attracteda continual interest of researchers in the past few decade. Nanocompositeoleogels of groundnut oil and stearic acid containing different percentagesof GO were developed to improve corneal permeation of ciprofloxacinHCl (CPH), an antibacterial drug, by Hasda et al.124 The in vitro release study showed thatby increasing the GO content up to 0.05% in the nanocomposite oleogels,a higher cumulative percentage of drug permeation through caprinecornea was seen. Moreover, by incorporation of GO in the oleogels,a higher ex vivo corneal permeation of CPH by Fickiandiffusion model was obtained.Recently,graphene and GO exhibited compatible physicochemical properties, whichmakes them suitable for biomedical applications, such as drug andgene delivery, biosensing and imaging, and tissue engineering.125 Silicon contact lensescontaining HA and rGO were prepared through direct loading of HA andrGO in the contact lens (HA-GO-DL) and also by the conventional soakingmethod (HA-GO-SM). The contact lenses containing lower amounts rGOexhibited acceptable swelling and transmittance properties. In addition,the HA-GO-DL contact lenses demonstrated a water retention propertyaccording to the water evaporation study results. Moreover, the fluxdata revealed that the HA-GO-DL lenses showed low burst with sustainedrelease up to 96 h, whereas a high burst release was seen for HA-GO-SMlenses after 24 h. Furthermore, the results of ocular irritation studyconfirmed the safety of the HA-GO-DL lenses. In comparison to HA-GO-SMand eye drop solution, a high HA-tear fluid concentration was seenby using the HA-GO-DL batch as well as improvement in the fluid volumeof rabbit tears.To treat and manage various cornealdiseases such as keratoconus and dry eye syndrome, eye drop solutionis widely used. However, due to a frequent dosing schedule, it canaffect the routine lifestyle of patients. In one study, hyaluronicacid (HA) and rGO loaded silicon contact lens were developed for cornealepithelial healing, which has the potential for improving the tearfluid volume for controlling numerous ocular diseases, such as dryeye syndrome.126 Owing to the very limited therapeutic efficacy of customarytreatments, such as eye drops and probable intraocular injection sideeffects, hydrogel contact lenses have been considered as suitableocular drug delivery systems due to their comfortable structure anddrug loading capacity. Considering this, in one research study, quaternizedchitosan (HTCC), silver nanoparticles, and GO were used for developmentof hydrogel-based contact lens, which had both antibacterial and antifungalactivities.127Recently, an active targeted drug deliveryvia endocytosis mediatedby ligand–receptors was used for doxorubicin (DOX) deliveryto choroidal melanoma. Transferrin (Tf)-modified pegylated graphenenanocarrier was used for DOX loading and provided a targeted capabilitytoward ocular choroidal melanoma-1 cells, which typically exhibiteda high expression of Tf receptors. DOX was loaded into the nanocarrierthrough π–π stacking architecture. The resultsdisplayed more than 80% tumor cell inhibition, which confirmed thepotential of using Tf-PG-DOX as an extremely effective antitumor drugdelivery system suitable for choroidal melanoma.128Bioinspired compound eyes (BCEs) as micro-optical devicesare tremendouslyinteresting due to their large fields of view (FOV) and various focalcapability. Wang et al. prepared a glycerol/graphene nanosheets (G/GNSs)-basedBCEs through a template-directed self-assembly process 9. For li129One-step laser reducedGO conductive tracks on transparent andflexible poly substrates also showed increasedabsorption toward a shorter wavelength of up to 96% in UV regions,which can significantly protect human eyes from high-energy lighthazards.4.2130 Hence, researchers have developed contact lenses usingelectronic devices for detecting physiological changes for the diagnosisof diseases.131 To develop electronicson soft contact lenses, optical transparency, stretchability, flexibility,and reliability upon repeated eye-blinks for clear vision are theimportant demanding challenges. To overcome these challenges, theycan be worn and produced by transparent, stretchable materials andharmless to the human body. Regarding the development of wearablesoft contact lenses, graphene and its hybrid with metal nanowireswere used as multifunctional sensors with suitable transparency andstretchability for wireless diagnosis and management of diabetes andIOP. Indeed, graphene and its hybrid provided enough transparencyand stretchability, which makes sure for the users that the soft contactlens is reliable and comfortable for unobstructed vision when it isworn.132 Recently, flexibility and transparencyof graphene led to development of liquid contact lenses with a largeFOV, a compact size, and fast response to electric potential.133Currently,contact lenses are used for cosmetic reasons and correction of vision.A unique platform can be provided for ocular diagnostics through continuouscontact of contact lenses with our tear fluids.134 High IOP causes loss of peripheral visual fieldsand leads to irreversible loss of vision fields.135 Unfortunately, glaucoma caused by high IOP is the secondmain cause of blindness in the world. As a result, measuring or monitoringthe IOP is important for glaucoma inhibition and management. Up tonow, many tonometers, the main tool to measure IOP, have also beendeveloped; however, they can be used by professionals for repetitivemeasurements to identify spikes and fluctuations of the patients’daily IOP.137 Zeng et al. developed an IOPsensor using transparent graphene to prevent the sensors from blockingvision and enhancing the flexibility of the wearable sensors to fitseveral sizes or curvatures in the eye.138 Electrooculography (EOG) is a technique to record the cornea-retinalstanding potential induced by eyeball movements. EOG applicationsare an ophthalmological diagnosis and can be used for developing wearablemedical sensors and as human–computer interaction interfaces.140 To resolve the limitation of silver/silver chloride (Ag/AgCl) gel-based,“wet” electrodes, a common method to measure the biopotentials,the graphene-coated textile electrodes were developed for monitoringcardiac biopotentials. Graphene textile electrodes with a high degreeof flexibility and stretchability can be used in various kinds ofpersonal clothing to monitor the epileptic patients and driver drowsiness,diagnostic polysomnogram tests for sleep disorders, and for developingwearable human–computer interfaces.141 Regarding the importance of visual electrophysiology measurements,soft graphene contact lens electrodes were used for conformal, fullcornea recording of electroretinography (ERG) from cynomolgus monkeys.ERG is a test to measure the electrical potential changes at the cornealsurface, which is employed in ophthalmic diagnostic testing for theassessment of retina functional integrity. The softness and opticaltransparency of graphene increased the high-efficacy measurementsof various kinds of ERG signals, including full-field ERG, multifocalERGs, and multielectrode ERG, with negligible corneal irritation.142 Besides, electrical signals in mouse retinawere investigated using a combination of field-effect transistorscontaining graphene and scanning photocurrent microscopy with microfluidicplatforms to detect the neural activity of retina. Results showedthat graphene concentration in the carrier can be modulated by electricalactivity in living retinal tissues, leading to potential gradientsthat can separate photoexcited electron–hole pairs and producephotocurrent signals.143Ocular hypertension is the most importantrisk factor in glaucoma, which has a higher IOP than normal.144 In this study, FLG was used fordevelopment of a sensor with high transparency, sensitivity, linearity,stability, durability, reliability, and biocompatibility for 24 hmonitoring of IOP and parylene-containing rGO and CNT. The sensing performance of preparedcontact lens sensor showed a high sensitivity of 36.01 μV mmHg–1 using an eyeball model made of PDMS to simulate thecurved surface of human eye. High sensitivity to IOP change, goodlinearity, good accuracy and great stability within the clinicallyrelevant IOP range were the outstanding properties of the fabricatedsensor. In another study, a contact lens for detection of IOP wasdeveloped using three-dimensional graphene nanowalls (GNWs) throughthe gold-assisted transfer method. The resistance response of thedeveloped sensor to the normal IOP fluctuation was 1.014 kΩmmHg–1 with a normal sensitivity of 42,250 ppm mmHg–1 and the response range of 0–75 mmHg accordingto the simulated tests on porcine eyes in vitro.The obtained results revealed that the GNWs have significant potentialfor continuous IOP monitoring with high sensitivity and low powerconsumption.146In addition, recently, a wearable contact lens sensor for noninvasivein situ monitoring of IOP was developed by Fan et al.,147 to monitor the IOP  were also employed in contact lensesby Zhang et al. the IOP 11. The G148 In general, it is crucially importantfor diabetic patients to detect their glucose sugar level for earlytreatment, so the noninvasive and real-time detection from tears willbe very valuable. In this study, a high-performance intraocular biosensorcontaining nitrogen-doped graphene was developed, which showed a highelectroactive property that can act as an ophthalmic electrode. Thefabricated chitosan-graphene based biosensor showed a high sensitivityat 9.7 μA mM–1 cm–2, a broadlinear range at 12 mM, and a good detection limit of 9.5 μM.The synthesized graphene-based biosensor also remained stable aftera month of storage. The in vitro ocular biocompatibilityof GC-COOH was investigated using CECs and RPE cells. The as-preparedGC-COOH was highly biocompatible to ophthalmologic cells. Moreover,the effect of biosensing electrode was examined on ocular tissues in vivo, and to monitor the intraocular blood sugar in tears,the electrode was evaluated as an assembled wearable corneal contactelectrode using New Zealand white rabbits as animal models  was fabricated for detection of glucose sugar fromtears.l models 12.148 Ac5124 while othersreported unfavorable biological responses and cytotoxicity.90 These inconsistent results might have been caused by differencesin research groups, experimental models/animals, and physicochemicalcharacterizations of GFNs and their compositions. Once GFNs are preparedand selected for ocular applications, their biocompatibility shouldbe evaluated, and further detailed and accurate experiments regardingtoxicity of GFNs must be done.Over the past few decades, GFNs have been exploredfor ocular therapeuticand diagnosis applications. The high specific surface area and largeπ-conjugated aromatic structure of GFNs make them good candidatesfor the development of ocular materials and devices, such as oculardrug delivery systems and sensors. Besides their advantages, thereare some challenges regarding GFNs’ toxicity especially forclinical use. Currently, since the literature about ocular toxicityof GFNs is limited, it is hard to conclude the potential GFNs’ocular hazards. Until now, there are two opposite opinions in thisresearch field: Some researchers suggested that GFNs are biocompatibleand can be good candidates for ocular applications,In most of the studies detailedherein, the toxicity of GFNs extremelydepends on their physicochemical properties, including size, surfacefunctional groups, oxidative state, and dose of administration aswell as exposure time. GFNs are very large family with huge differencesin size and dimension, which affect the toxicity. In addition, studiesfrequently showed that unmodified graphene and GO were more cytotoxiccompared with functionalized GFNs and rGO. Therefore, useful surfacemodification must be carefully evaluated and used to decrease theGFNs’ cytotoxicity for ocular applications in the future. Surfacefunctionalization of GFNs using biopolymers, such as PEG, can ledto an increase their biocompatibility in ocular applications and improvetheir therapeutic effects. Another physicochemical property that caninfluence the ocular toxicity of GFNs is degree of oxidation. A promisingapproach for reducing the GFNs’ ocular toxicity and improvingtheir safety for ocular applications is minimizing the degree of oxidation.In addition, another important issue that needs to be consideredis the long-term fate of GFNs after entering the eye or being takenup by cells. In most of the reported studies, the short-term oculartoxicity has been assessed, but long-term follow-up period shouldbe considered. Therefore, extended research is needed to evaluatewhether longer treatment times can affect the toxicity of GFNs inocular applications or not.To improve the safety of using GFNsin ocular applications, itis essential to study their toxicity mechanisms. To date, variousocular toxicity mechanisms of GFNs have been investigated and extensivelyapproved, including mitochondrial damage, oxidative stress, inflammatoryresponse, apoptosis, necrosis, cell membrane damage, cell death, cellcycle disorder, and cell viability loss. However, more specific pathwaysof the ocular toxicity mechanism of GFNs need to be discovered andinvestigated. For a better understanding of complex interactions betweenGFNs and biological systems, gene expression profiles should be studiedas an important approach, which reveals the potential molecular mechanismsof toxicity on a whole cell level.Moreover, hydrogels containingGFNs have been extensively employedand studied in contact lenses since they are highly comfortable andbiocompatible and have a high surface area. However, there will besome drawbacks by changing some conditions, such as increasing thecontent of GFNs and time of exposure. In addition, selecting the rightcell lines and/or animal model in assessment of ocular toxicity iscrucially important to develop ocular formulations with proper safetyand efficiency.in vitro and in vivo ocular toxicities must be evaluated. Therefore,GFNs’ biocompatibility, stability, and biological performancesas well as their side effects in ocular applications can be preliminarilyobtained by considering these items. However, future research is necessaryfor exploring the biological responses and the safety issues of GFNsby taking into consideration the different physicochemical properties.Before doing any research, personal safety protection is cruciallyneeded when dealing with GFNs both in production and in a researchenvironment. All of these provided results will further improve therequired knowledge for developing safe technologies and products usingGFNs appropriate for biomedical applications and minimizing the risksto human health. Consequently, further research is still necessaryto overcome the aggregation problems and genotoxicity of GFNs as wellas their toxicity dependency on the size- and/or functional groupsbefore we can draw a conclusion whether GFNs are safe or not.In conclusion, since ocular devices based onGFNs are developing,for detailed and accurate information about the interactions of GFNsat the molecular, cellular, and tissue levels, their physicochemicalproperties as well as their