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Graphene oxide (GO) has several potential biomedical applications and therefore cytotoxic evaluation of GO is very important. However, the two most common in vitro models for testing cytotoxicity—primary human cells and immortalized cell lines—suffer serious limitations, namely limited supplies of cells and unrealistic cellular responses, respectively. Here, we demonstrate the use of embryonic stem cell (ESC)‐derived cells to study GO cytotoxicity. We tested the use of retinal pigment epithelium (RPE) cells derived from three‐dimensional human ESC cultures (“ESC‐RPE” cells) as a model of GO cytotoxicity by exposing them to varying concentrations of GO nanosheets. For comparison, we also performed the same test with primary human retinal pigment epithelium cells (“hRPE”), and with cells derived from a human RPE cell line (“ARPE19” cells). We found that cytotoxicity metrics (viability, apoptosis, intracellular reactive oxygen species, and mitochondrial membrane potential) were very similar in ESC‐RPE cells and hRPE cells, and those in ARPE19 cells were very different. We conclude that cell models of GO cytotoxicity derived from ESCs are an excellent alternative to primary human cells, without the limitations of tissue availability.

Rationale:Discriminating between aglycone-substituted and saccharide-substituted saikosaponins by liquid chromatography/tandem mass spectrometry (LC/MS n ) is a long-standing issue that is still to be resolved. It is necessary to characterize the two types of substituted saikosaponins taking into consideration the potential significant difference in their bioactivity. Methods:Taking the substituents malonyl and acetyl as examples, we developed a MS strategy to discriminate between the aglycone-substituted and saccharidesubstituted saikosaponins through comparing their Y 0 − nH 2 O (n = 1-2) ions from the protonated molecules in the full-scan mass spectra and their B ions in the MS 2 spectra of sodium-adduct molecules in the positive mode. Results:The deprotonated molecules of the aglycone-substituted saikosaponins presented similar fragmentation patterns to those of saccharide-substituted ones in the negative mode, which could not discriminate whether the substitutes were located on the aglycone or the saccharide. In contrast, the Y 0 − nH 2 O (n = 1-2) ions containing or no substituent were observed respectively in the mass fragmentation of the protonated molecules of aglycone-substituted or saccharide-substituted saikosaponins in the positive mode. In addition, the B ions containing or no substituent were observed respectively in the mass fragmentation of the sodiumadduct molecules of the saccharide-substituted or aglycone-substituted saikosaponins in the positive mode. Two aglycone-malonylated saikosaponins were reported for the first time. Conclusions:Whether the substituents were located on the aglycone or the saccharide could be determined according to the Y 0 − nH 2 O (n = 1-2) ions from the protonated molecules in the full-scan mass spectra and the B ions in the MS 2 spectra of sodium-adduct molecules in the positive mode. Our results have updated the mass fragmentation patterns of substituted saikosaponins, which is helpful for the quality control of pharmaceutical preparations containing saikosaponins. More importantly, this MS strategy should be able to be extended to characterize other substituted saponins of bioactive significance in future studies.

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The incorporation of functional groups endows graphene oxide (GO) with different surface charges, which plays important roles in biological interactions with cells. However, the effect of surface charge of GO derivatives on ocular biocompatibility has not been fully elucidated. Previously, we found that positively, negatively and neutrally charged PEGylated GO (PEG-GO) nanosheets exerted similar effect on the viability of ocular cells. In this work, we performed in vitro and in vivo studies to comprehensively study the effect of surface charge of PEG-GO on ocular compatibility. The in vitro results showed that the cellular uptake efficacy of negatively charged PEG-GO nanosheets was significantly decreased compared with positively charged and neutrally charged analogs. However, three kinds of PEG-GO nanosheets produced similar amounts of intracellular reactive oxygen species and showed similar influence on mitochondrial membrane potential. By analysis of global gene expression profiles, we found that the correlation coefficients between three kinds of PEG-GO-treated cells were more than 0.98. Furthermore, in vivo results showed that all these PEG-GO nanosheets had no significant toxicity to ocular structure and function. Taken together, our work suggested that surface charge of PEG-GO exerted negligible effect on its ocular compatibility, except for the cellular uptake. Our work is conducive to understanding the relationship between surface charge and biocompatibility of GO derivatives.