www.acsanm.org Article Effects of Lateral Size, Thickness, and Stabilizer Concentration on the Cytotoxicity of Defect-Free Graphene Nanosheets: Implications for Biological Applications Chen-Xia Hu,+ Oliver Read,+ Yuyoung Shin, Yingxian Chen, Jingjing Wang, Matthew Boyes, Niting Zeng, Adyasha Panigrahi, Kostas Kostarelos, Igor Larrosa, Sandra Vranic,* and Cinzia Casiraghi* Cite This: ACS Appl. Nano Mater. 2022, 5, 12626−12636 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: In this work, we apply liquid cascade centrifugation to highly concentrated graphene dispersions produced by liquid- phase exfoliation in water with an insoluble bis-pyrene stabilizer to obtain fractions containing nanosheets with different lateral size distributions. The concentration, stability, size, thickness, and the cytotoxicity profile are studied as a function of the initial stabilizer concentration for each fraction. Our results show that there is a critical initial amount of stabilizer (0.4 mg/mL) above which the dispersions show reduced concentration, stability, and biocompat- ibility, no matter the lateral size of the flakes. KEYWORDS: graphene, liquid-phase exfoliation, pyrene, biocompatibility, atomic force microscopy, size-thickness characterization 1. INTRODUCTION traditional pyrene derivative stabilizers due to the presence Two-dimensional (2D) materials have drawn increasing of two pyrene cores functionalized and linked by a single amounts of attention in recent years due to their unique pyrrolidone central group. Despite the molecule being water- properties.1−4 To apply 2D materials in practical applications, insoluble, we observe enhanced exfoliation efficiency in low-cost and large-scale synthesis approaches are needed.4 comparison to mono pyrene stabilizers such as 1-pyrenesul- Among them, liquid-phase exfoliation (LPE) is an effective and fonic acid sodium salt (PS1), which is soluble in water.14 This scalable method to produce dispersions of 2D materials.5,6 was attributed to the higher interaction strength between BPS This approach is most commonly performed using organic and graphene due to the presence of two pyrene groups (rather solvents;5,6 however, it can be extended to water with the than just one as in PS1) and the insolubility of the molecule, assistance of suitable stabilizing molecules, enabling us to i.e., the BPS molecule prefers to adsorb onto graphene rather target biological applications including imaging and drug than interacting with water molecules, hence improving the delivery.7,8 exfoliation efficiency.14 When the molecule is adsorbed onto Non-covalent functionalization of 2D materials with graphene, it prevents reaggregation of the exfoliated nano- dispersing agents facilitates the exfoliation and stabilization sheets in solution via electrostatic stabilization, provided by the of 2D materials in water, leading to highly concentrated and charged functional group, similar to stabilization with ionic stable dispersions.9−11 This simple supramolecular approach surfactants.20 In this work, we exploit the highly concentrated enables us to tune the exfoliation yield, surface charge, and graphene dispersions produced in water using the BPS chemistry by simply selecting different types of disper- stabilizer to fraction the nanosheets into narrow size and sants.12−20 Among them, small aromatic molecules and, in thickness distributions by liquid cascade centrifugation particular, pyrene derivatives, show very effective exfoliation (LCC).22 This enables us to understand how the initial and stabilization efficiency for graphene in water when amount of stabilizer and the size and thickness distributions of compared to conventional surfactants and polymers.16,21 However, the exact exfoliation efficiency and dispersion stability strongly depend on the precise structure of the pyrene Received: June 1, 2022 stabilizer,14,15,17,18 whereas the size and thickness of obtained Accepted: August 25, 2022 flakes are largely unaffected by the type of pyrene derivative.13 Published: September 12, 2022 Recently, our group has reported the use of a new pyrene derivative, called bis-pyrene (BPS) (Figure 1). The molecular structure of the BPS molecule differs from the one of © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsanm.2c02403 12626 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article Figure 1. Schematic of the liquid-phase exfoliation of graphite and liquid cascade centrifugation to produce size-selected fractions A, B, and C from a stock dispersion produced with a fixed amount of BPS stabilizer. the graphene nanosheets affect the biocompatibility of these subtraction was used with a DI water baseline. Dilutions of the BPS- dispersions. stabilized graphene fractions were prepared using DI water, and Here, we show a systematic analysis on the cytotoxicity of measurements were taken in UV-cuvettes with a path length of 1 cm. defect-free graphene dispersions produced by exfoliation in The concentration was calculated by using an absorption coefficient at water using BPS. We demonstrate that LCC can be used to 660 nm 2460 L g−1 m−1,5 by using the Beer−Lambert law.23 2.4. Zeta-Potential Measurements. A ZetaSizer Nano ZS fraction the graphene dispersions into stable and concentrated purchased from Malvern Instruments, UK was used to obtain the dispersions (concentration > 0.5 mg/mL) with nanosheets electrophoretic mobility (μ). Dispersions were diluted with DI water with narrow lateral size and thickness distributions, which do and placed in a folded capillary cell for analysis following a default not depend on the initial stabilizer amount. Dose-escalation instrument setting at 25 °C and at the natural pH value. In this studies performed in the human epithelial bronchial equipment, zeta-potential (ζ) values were converted from μ by using immortalized cell line (BEAS-2B) demonstrated that the Henry’s equation.24 All samples were measured three times, and the cytotoxicity profile strongly depends on the initial amount of final ζ values were calculated and quoted as mean ± standard stabilizer: below 0.4 mg/mL, the cytotoxicity is affected by the deviation (SD). lateral size of the nanosheet, with the largest nanosheets (>200 2.5. Dynamic Light Scattering (DLS) Measurements. A nm) showing good biocompatibility (up to 75−100 μg/mL ZetaSizer Nano ZS purchased from Malvern Instruments, UK was dose). Above a 0.4 mg/mL stabilizer concentration, the used to obtain the hydrodynamic size of graphene flakes. Dispersions were diluted with DI water and placed in disposable polystyrene cytotoxicity does not show any dependence on the lateral size: cuvettes (Malvern Instruments, UK) for analysis following a default all fractions show reduced biocompatibility, as compared to the instrument setting at 25 °C and at the natural pH value. The intensity ones obtained with lower stabilizer concentrations. obtained for each test was used for calculation of Z-average size values after cumulated analysis.25 Although these hydrodynamic size values 2. EXPERIMENTAL SECTION yielded by DLS measurements were not designed for 2D materials, 2.1. Materials. Graphite flakes (99.5% grade) were purchased these values can be used to confirm the size evolution during LCC from Graphexel Ltd. Silicon wafers (Si/SiO2 with an oxide layer fractionation.26 All samples were measured three times, and the mean thickness ∼290 nm) were purchased from Inseto Ltd. (UK). BPS was of the final Z-average values was calculated. synthesized in house as outlined in a previous work.14 Deionized 2.6. AFM Characterization. A Bruker MultiMode 8 atomic force water was dispensed from a Millipore Simplicity 185 water microscope in PFT with ScanAsyst mode, equipped with ScanAsyst- purification system. Isopropanol (IPA) was purchased from Sigma- Air cantilevers, was used for all AFM measurements. Aliquots (10 μL) Aldrich. of each fraction were drop cast onto clean, pre-heated (150 °C), Si/ 2.2. Preparation of Graphene Dispersions. A Hilsonic bath SiO2 wafers (0.5 cm2), washed with DI water and IPA, and then sonicator (600 W, 30 kHz) with a chiller unit maintaining bath annealed at 250 °C for 2 h. AFM measurements were taken on each temperature at 10 °C was used for all exfoliation steps. A Sonorex RK sample, following the methodology outlined in detail in ref 13 and 100 bath sonicator (140 W, 35 kHz) was used for substrate cleaning collecting multiple maps capturing over 150 isolated flakes for each and redispersion of exfoliation material during LCC. A Sigma 1-14k fraction. The morphological parameters, lateral size (L) and apparent refrigerated centrifuge, with a 12084 rotor and 2.5 mL Eppendorf thickness (T), were then extracted automatically from the maps using vials, was used for all centrifugation steps. BPS-stabilized graphene the Gwyddion software, as described in ref 13. dispersions were produced via LPE in water following the method- 2.7. Raman Spectroscopy. A Renishaw Invia Raman spectrom- ology outlined in our previous work.14 eter was employed for Raman measurements. A laser at 514.5 nm with 2.3. UV−Vis Spectroscopy. A PerkinElmer l-900 UV−vis−NIR 2.0 mW laser power was used for all measurements. We measured spectrophotometer was used to measure the UV−vis spectrum of 40−50 isolated flakes, drop cast onto Si/SiO2 substrates, for each graphene dispersions between 250 and 800 nm. Automatic baseline fraction. A 100× (NA = 0.85) objective lens and a 2400 grooves/mm 12627 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article grating were used. The qualitative thickness distribution of the 3. RESULTS AND DISCUSSION graphene nanosheets was extracted by fitting the 2D peak of each spectrum with a Lorentzian line shape. The coefficient of 3.1. Size and Thickness Characterization. In our initial determination, R2, is then used to discriminate between single-layer study using the BPS stabilizer,14 we have shown that the initial graphene (SLG) with N = 1 (associated to R2 = 0.99−1.00), few-layer graphene (FLG) with 1 > N < 7 (associated to R2 = 0.97−0.98), and bulk graphite with N > 10 (associated to R2 = 0.95−0.96). A symmetric 2D peak corresponds to SLG, an asymmetric 2D peak corresponds to FLG, and two peaks correspond to bulk graphite. This qualitative method enables us to extract thickness distributions from the Raman spectra, as described in more detail in previous works.27−31 2.8. Thermogravimetric Analysis (TGA). TGA experiments were carried out on a TA Instruments SDT-650. Measurements were made in a nitrogen atmosphere with a heating rate of 10 °C/min from room temperature to 800 °C. To isolate the graphene from the dispersions, vacuum filtration was utilized to produce membranes of the material. These membranes were then dried under vacuum at 250 °C and torn for the TGA experiments. 2.9. Cell Culture. The human epithelial bronchial immortalized cell line (BEAS-2B, CRL-9609TM, ATCC, and LGC standards, UK) was maintained in an RPMI 1640 cell culture medium (Sigma- Aldrich, Merck Sigma, UK) and supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, UK) and 1% penicillin−streptomycin (Sigma-Aldrich, Merck Sigma, UK) at 37 °C in a humidified 5% CO2 incubator. Cells were passaged twice a week using a 0.05% Trypsin−EDTA solution (Sigma-Aldrich, Merck Sigma, UK) when reaching 80% confluence. The activity of trypsin was stopped using 10% FBS. 2.10. Cell Treatments. Cells were seeded in 12-well plates (Corning, Costar, Sigma-Aldrich, Merck Sigma, UK) for toxicity assessment performed using optical microscopy and flow cytometry or in CellviewTM cell dishes (627870, Greiner Bio-One Ltd., UK) for uptake experiments, using confocal microscopy. Cells were seeded and treated (when reaching 60−80% confluence) in a complete cell culture medium (see the Cell Culture section), unless stated Figure 2. (a) Concentration calculated using the Beer-Lambert law otherwise. and UV−vis spectroscopy, (b) zeta potential calculated by conversion 2.11. Optical Imaging and Flow Cytometry. Cells were treated from μ using Henry’s law and DLS measurements, and (c) with GR0.3A/B/C, GR0.4A/B/C, GR0.6A/B/C, and GR1.0A/B/C hydrodynamic size from DLS measurements of the A, B, and C (at graphene concentrations of 25, 50, 75, and 100 μg/mL, 1 mL/ fractions obtained by LCC made with different initial BPS stabilizer well, respectively) in serum-free RPMI for the initial 4 h. After 4 h of amounts (0.3, 0.4, 0.6, and 1 mg/mL). incubation, FBS (100 μL/well) was added to the cells and the cells were further incubated for another 20 h. After 20 h of incubation, amount of BPS affects the final graphene concentration. In images were taken using an EVOS FL microscope (10× objective, particular, dispersions produced using an initial amount of BPS transmitted channel). GR0.3 and GR0.4 treated cells were detached using Trypsin−EDTA (300 μL/well, 5 min), neutralized with FBS less than 0.3 mg/mL resulted in the lowest graphene (30 μL/well), centrifuged (1500 rpm, 5 min), resuspended in 1× concentrations.14 Therefore, for this study, we prepared four diluted annexin-binding buffer (200 μL/sample, Molecular Probes, graphene dispersions using different initial amounts of BPS Thermo Fisher Scientific, UK), and stained with annexin V Alexa equivalent to 0.3, 0.4, 0.6, and 1.0 mg/mL water solvent. The Fluor 488 (AV) in the dark (1 μL/sample, 20 min, room temperature, corresponding samples were named GR0.3, GR0.4, GR0.6, and Thermo Fisher Scientific, UK). The samples were stored in ice, and GR1.0, respectively. In detail, graphite (3 mg/mL), BPS (0.3, propidium iodide (PI, 1 μL/sample, Sigma-Aldrich, Merck Sigma, 0.4, 0.6, and 1.0 mg/mL), and DI water (100 mL) were added UK) was added shortly before analysis. A population of 10,000 cells to a reagent bottle (250 mL) and sonicated for 7 days was analyzed on a BD FACSVerseTM flow cytometer with 488 nm continuously. The unexfoliated graphite was removed by excitation. Band-pass filters (515 and 615 nm) were used for annexin V and propidium iodide detection, respectively. centrifugation at 3500 rpm (903 g) for 20 min, which was 2.12. Confocal Microscopy. Cells were treated with GR0.3A/C repeated twice. This step was used to sediment a large amount (graphene concentration = 25 μg/mL, 0.5 mL/well), GR0.6A/C of the residual unexfoliated graphite from the stock dispersion, (graphene concentration = 10 μg/mL, 0.5 mL/well), and GR1.0A/C which was then discarded, while the supernatant was retained. (graphene concentration = 10 μg/mL, 0.5 mL/well) for 24 h. Cells LCC was then used to separate the resulting supernatant into were washed (RPMI culture medium with 10% FBS, 0.5 mL/well, fractions containing flakes with differing distributions in lateral ×2) and incubated in CellTrackerTM Green dye, a green 5- size and thickness. The following cascade was used for each chloromethylfluorescein diacetate (CMFDA) containing solution (3 dispersion: 5000 rpm (1844 g) for 30 min, 9000 rpm (5976 g) μM, 0.5 mL/well, 15 min, diluted in RPMI culture medium with 10% for 30 min, and 15,000 rpm (16,603 g) for 60 min. For each FBS, C7025, Thermo Fisher Scientific, UK). After incubation, the dispersion, prepared with a fixed amount of BPS, we obtained CellTrackerTM Green CMFDA dye-containing solution was removed and replaced by the RPMI culture medium with 10% FBS (0.5 mL/ three sub-sets of samples, denoted with A, B, and C, e.g., well). Cells were then examined under a Zeiss 780 confocal laser GR0.3A, GR0.3B, and GR0.3C, respectively. Figure 1 shows a scanning microscope using a 40× objective. The confocal images were schematic of the LPE of graphite and the following LCC processed by the Zeiss microscope software ZEN. Excitation/ methodology that was employed in this work. LCC is expected emission wavelength: FDA = 492/517 nm. to result in the sedimentation of heavier and larger flakes at 12628 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article Figure 3. Distributions of (a) lateral size and (b) apparent thickness for GR0.3, GR0.4, GR0.6, and GR1.0 datasets, as measured by AFM. The whiskers show the min/max values, the black square shows the mean, the box shows the interquartile range, and the horizontal line in the box shows the median. (c) Average lateral size vs average thickness for all samples and fractions with error bars showing one standard deviation from the mean and β the gradient of the linear fit. The error bars show the SD in lateral size and apparent thickness. tration of the GR1.0 fraction is significantly lower. These results may suggest that a lower initial concentration of the BPS stabilizer is favorable to produce highly concentrated dispersions. This facilitates the isolation of fractions containing higher concentrations of smaller and thinner flakes from the stock dispersion, i.e., the higher the concentration of the stock dispersion, the higher the concentration of smaller and thinner flakes within the stock dispersion. Figure 2B shows the recorded zeta potential of the three fractions from each dispersion: besides fraction C of GR1.0, the zeta potential is always higher than 30 mV, which is the threshold (in absolute value) above which a graphene colloidal suspension is considered stable.32 We note a similar rough decreasing trend of zeta potential with increasing initial BPS concentration, which may indicate that the excess stabilizer has a detrimental effect on the dispersion stability. Figure 4. Single-layer graphene (N = 1), few-layer graphene (1 > N < Figure 2C shows the hydrodynamic size of each fraction 7), and graphite (N > 10) percentages of each fraction from the four obtained by DLS, revealing a clear trend of decreasing dispersions, extracted from the qualitative analysis of the Raman hydrodynamic size with each subsequent fraction obtained spectra. by centrifugation at higher forces. Additionally, we note that the hydrodynamic size of each fraction is roughly similar lower centrifugal forces and smaller and thinner flakes at higher regardless of the initial BPS concentration. Although DLS centrifugal forces.22 Each fraction should therefore contain provides only qualitative results, these measurements confirm flakes of a range of sizes and thicknesses that sediment between that the LCC methodology has effectively separated the stock the higher and lower centrifugal forces used in each step of the dispersion into fractions with decreasing hydrodynamic size. cascade. TGA was used to analyze the BPS amount in selected To gain understanding of the exfoliation efficiency and graphene dispersions: a higher amount of stabilizer used to stability of each graphene fraction, the concentration, zeta prepare the dispersion does not necessarily result in a higher potential, and hydrodynamic size of each fraction were amount of stabilizer adsorbed on the nanosheets as sample measured (Figure 2). Note that the concentration and stability preparation involves different steps such as centrifugation and are in particular very important to perform accurate biological washing, in which molecules may get removed. For simplicity, studies.15 we selected the GR0.3 and GR0.6 dispersions for TGA Figure 2A shows that the concentration of dispersed material analysis. Supporting Information, Figure S1 shows the TGA reduces with each subsequent fraction. In all samples, however, thermograms for the BPS powder and membranes made from the concentration remains above 0.5 mg/mL, hence enabling the two selected dispersions. Full details of the thermograms further biological characterization (Section 3.2). Fractions A of are provided in the Supporting Information. For both graphene the GR0.3 and GR0.4 dispersions are extremely concentrated dispersions, the mass starts to decrease after 200 °C. There is at ∼5.2 mg/mL, which is significantly higher than the then a steady decrease in mass until 500 °C where the profiles corresponding GR0.6A and GR1.0A fractions, which have level off up to the final temperature of 800 °C. We can see that concentrations of 1.9 and 1.6 mg/mL, respectively. We note the loss of mass due to the BPS is much higher in the GR0.6 that the dispersion concentration of fraction B roughly sample as compared to the GR0.3, with the total mass lost for decreases with increasing initial BPS concentration, with the the GR0.6 sample being 32.3% and the mass lost for the GR0.3 GR0.3 having the highest concentration, GR0.4 and GR0.6 sample being 23.4%, indicating a higher amount of BPS in the having comparable but lower concentrations, and GR1.0 former sample. It should be noted here that due to the TGA having the lowest. The concentration of fraction C is roughly experiments being performed under a nitrogen atmosphere, similar for GR0.3, GR0.4, and GR0.6, whereas the concen- little-to-no mass loss can be attributed to the loss of carbon 12629 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article Figure 5. Cytotoxicity of defect-free graphene produced by LPE with BPS in BEAS-2B cells after 24 h of treatment (in the absence of serum for the first 4 h) using different dose concentrations (25, 50, 75, and 100 μg/mL) of (a) GR0.3A/B/C and (b) GR0.4A/B/C, assessed by optical imaging. Images outlined in red indicate the lowest dose concentrations in which cellular stress was observed for each fraction of graphene nanosheets. Scale bars = 100 μm. from graphene. Hence, in the case of BPS, a higher amount of there is likely a significant difference between the distributions stabilizer used during exfoliation translates to a higher number of flake thicknesses within the two dispersions, and therefore, of molecules adsorbed on the nanosheets. we have achieved successful fractioning by flake thickness. A A statistical AFM methodology13,33−34 was then employed larger overlap of interquartile range was observed between to collect a large, representative sample of lateral size and fractions for the GR0.6 and GR1.0 dispersions, for GR0.6, thickness measurements of flakes from fractions from each particularly between fractions B and C, and for GR1.0, dispersion, which was then used to evaluate the distribution particularly between fractions A and B, with the median of and to calculate the mean average lateral size (⟨L⟩) and mean each within the interquartile range of both fractions. This apparent thickness parameters (⟨T⟩), which are reported in the indicates that there is likely no significant difference between Supporting Information, Table S5. Figure 3A shows the lateral the thickness distributions of the two fractions. From each size distributions for each dispersion, which confirms a clear dispersion, fraction B contained flakes mostly <10 nm thick, decrease in mean and median lateral size with each subsequent which is indicative of FLG, whereas the thinnest flakes were fraction isolated at higher centrifugal forces. For GR0.3, GR0.4, typically found in fraction C with the majority of flakes having and GR1.0, there is no overlap between the interquartile ranges thickness < 5 nm. The minimum apparent thickness of any of each respective fraction, indicating that there is a significant flake recorded was ∼1 nm, which is likely indicative of SLG.19 difference between the flake size distributions of each fraction, In summary, the average flake layer number within the fraction and hence confirming successful fractioning by size. For sample decreases with increased centrifugal force, although the GR0.6, there is some overlap with the interquartile range of variations are statistically less relevant than those observed fractions B and C, with the median of each outside the range, with the lateral size. Hence, from our results, we can observe which may not only indicate that the fractions are statistically that LCC preferentially sediments and fractionates flakes via different but also suggests less discrete size selection in this lateral size more so than flake thickness. case. Figure 3C shows the plot of ⟨L⟩ vs ⟨T⟩ for each fraction Figure 3B shows the apparent thickness distributions for from the four dispersions: this reveals a log-normal trend each dispersion. A clear decrease in mean and median between size and thickness, which manifests itself as a linear thickness was observed with each subsequent fraction isolated trend on a log−log scale with a gradient of 1.2. This observed at higher centrifugal forces, in agreement with the trend in trend is in agreement with the results obtained with other lateral size (Figure 3A). There is a very small amount of pyrene-stabilized graphene dispersions as well as other overlap between the interquartile ranges of fractions A, B, and dispersant-assisted LPE dispersions.13,33 C of GR0.3 and GR0.4, with the median of each fraction We also calculated the ⟨L⟩ and ⟨T⟩ for the stock dispersion outside the interquartile range of each other. This suggests that by combining each individual dataset from fractions A, B, and 12630 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article Figure 6. Cytotoxicity of defect-free graphene produced by LPE with BPS in BEAS-2B cells after 24 h of treatment (in the absence of serum for the first 4 h) using different dose concentrations (25, 50, 75, and 100 μg/mL) of (a) GR0.6A/B/C and (b) GR1.0A/B/C, assessed by optical imaging. Images outlined in red indicate the lowest dose concentrations in which cellular stress was observed for each fraction of graphene nanosheets. Scale bars = 100 μm. C for each dispersion (Supporting Information, Table S5, group, to qualitatively identify and class the nanosheets as sample indicated as “ABC”). This is opposed to taking AFM SLG, FLG, or graphite.15,27−30 Figure 4 shows that all fractions measurements from samples of the stock dispersion, which contain between 2 and 20% SLG, 60 and 70% FLG, and 2 and would be both challenging and time consuming to do due to 25% graphite. Tabulated results from Raman spectroscopy can the broad range of sizes and thicknesses of flakes within the be found in the Supporting Information, Table S7. We note dispersion. On comparison of the stock dispersion, it appears that fraction C typically contains the lowest percentage of that the initial BPS concentration has no significant effect on graphite and the greatest percentages of SLG and FLG, the lateral size or apparent thickness distributions (Supporting whereas fractions A and B typically contain higher percentages Information, Figure S6), with an ⟨L⟩ of ∼200 nm and a ⟨T⟩ of of graphite due to the LCC process. Additionally, we tend to ∼7 nm recorded for each dispersion. observe a higher percentage of graphite within the dispersions These results indicate that the liquid cascade used in these produced using higher initial BPS concentrations (GR0.6 and experiments has successfully separated the stock dispersion GR1.0), which is in agreement with the lowest concentration into three discrete fractions by size and thickness, although reported for these samples (Figure 2). These findings are size-selection by centrifugation appears to be more successful broadly consistent with the AFM data when the apparent at separating the flakes via size than thickness. However, this thickness was converted into layer number (N) using may be a result of aggregation and restacking of flakes during methodology reported in ref 13 (Supporting Information, sample preparation or from contributions to the overall thickness by residual stabilizer molecules. The stability and Table S8 and Figure S9), but the percentage of SLG was concentration of the four dispersions produced and each size- significantly lower. selected fraction were adequate for subsequent biological In summary, we applied LCC to the stock dispersions, each testing. produced with a fixed amount of BPS stabilizer, obtaining In addition to the AFM measurements, we also employed fractions containing graphene nanosheets with narrow lateral Raman spectroscopy to characterize over 40 nanosheets from size and thickness distributions. Our results show that a lower each fraction. The Raman spectrum of the nanosheets shows initial amount of the BPS stabilizer produces stable dispersions the characteristic G and D peaks at ∼1580 and ∼1350 cm−1, in water with higher concentrations of graphene. By respectively, and the 2D peak at ∼2680 cm−1.35 The D peak is considering the stock dispersion, i.e., combined fractions A, observed in the spectra of graphene produced by LPE, B, and C, we confirm that the initial concentration of the BPS although in this specific case, the D peak is not attributed to stabilizer has no significant effect on the lateral size or structural defects but it is activated by the edges of the thickness of the nanosheets produced. By comparing results nanosheets, whose size is smaller than the laser spot.5,19,35,36 from both AFM and Raman studies, we show that the We used a method, developed and tested previously in our dispersions contain mostly FLG with <10% graphite in most 12631 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article cytotoxicity effect is induced in BEAS-2B cells in the absence of FBS.40 Herein, for the initial 4 h of exposure, cells were treated with the material without serum to maximize the interaction between graphene sheets and the cells. It is apparent from Figures 5 and 6 that defect-free graphene prepared using a lower initial amount of stabilizer is less cytotoxic to BEAS-2B cells compared to the ones prepared with higher initial amounts of stabilizer. For example, the lowest dose concentrations in which signs of cellular stress (e.g., reduced cell confluences, cell morphology alteration, and cell detachment) were observed for GR0.3A/B/C and GR0.4A/B/C are 75/75/50 and 100/100/75 μg/mL, respectively. Meanwhile, for GR0.6A/B/C and GR1.0A/B/C, the lowest dose concentration at which cellular stress was observed is at 25 μg/mL. We also observed increased cellular toxicity with smaller sized graphene. For example, in GR0.3 and GR0.4, the lowest toxic dose concentration is lower for graphene with the smallest size (C) compared to the larger ones (A and B). To validate the size-dependent effect of defect-free graphene produced by LPE with BPS, the cytotoxicity of GR0.3 and GR0.4 has been further assessed by PI/AV staining using flow cytometry (Figure 7). In agreement with the optical images, the result confirmed the size-dependent toxicity effect of GR0.3 and GR0.4, with smaller size graphene nanosheets causing greater cytotoxicity. For example, in Figure 7, we show that at the highest tested concentration of 100 μg/mL, cell viability (indicated by PI/AV double negative stained cells) reached 93.88% (96.01%) for GR0.3A (GR0.4A), decreased to 80.87% (88.86%) for GR0.3B (GR0.4B), and further lowered Figure 7. Cytotoxicity of defect-free graphene produced by LPE with to 0.82% (75.81%) for GR0.3C (GR0.4C). It is necessary to BPS in BEAS-2B cells after 24 h of treatment with (a) GR0.3A/B/C clarify that the lowest toxic dose concentration reported using and (b) GR0.4A/B/C (25, 50, 75, and 100 μg/mL), assessed by PI/ optical imaging refers to the lowest dose concentration at AV staining using flow cytometry. The PI/AV double negative stained which cellular stress is observed. However, cellular stress does cell percentages (PI-/AV- %) were reported. DMSO (20%) was used not equate to cell death, hence the discrepancy between visual as the positive control. inspection of cells by optical imaging and percentages of alive cells quantified by flow cytometry. cases and 10−20% SLG content, which is in agreement with To understand the potential underlying mechanism of the previous results on LPE with pyrene derivatives.13−15,17 toxicity induced by the material, the uptake profile for the 3.2. In Vitro Studies. Dimensionality, lateral size, surface three sets of graphene sheets (GR0.3, GR0.6, and GR1.0) was charge, and functionalization as well as chemical composition assessed in BEAS-2B cells by confocal imaging (see Figure 8). of the nanomaterial can lead to radically different interactions The uptake experiment is performed at sub-toxic dose with living systems.37 In the framework of 2D materials, concentrations (25 μg/mL for GR0.3, 10 μg/mL for GR0.6 graphene oxide (GO) is the most commonly used graphene and GR1.0). Furthermore, fraction B has been excluded in this derivative for biological applications.38 In vitro and in vivo experiment to be able to compare the graphene sheets with the effects of GO have been extensively studied.39 However, largest (A) and smallest size (C). As shown in Figure 8, uptake cytotoxicity studies of defect-free graphene obtained by non- of GR0.3A/C, GR0.6A/C, and GR1.0A/C was observed in covalent functionalization as a function of size and thickness BEAS-2B cells. In general, no obvious differences in the uptake are missing. amounts were observed between graphene fractions A and C. The cytotoxicity profile of defect-free graphene produced by BEAS-2B cells are labeled by the CMFDA dye, and in the LPE with BPS was first assessed via optical imaging of BEAS- presence of graphene internalization, the green intracellular 2B cells treated with the materials. We remark that cytotoxicity fluorescence signal is quenched by the black material. Co- tests on the BPS stabilizer alone cannot be performed as the localization of the material in the bright field channel and BPS molecule is insoluble in water.14 Figures 5 and 6 show quenched signal in the CMFDA channel indicates graphene optical images of BEAS-2B cells after 24 h of treatment with internalization, as clearly shown in Figure 8. The uptake of different concentrations (25, 50, 75, and 100 μg/mL) of graphene sheets suggests that the mechanism of cytotoxicity graphene sheets exfoliated with lower (GR0.3A/B/C and could have been triggered intracellularly. In fact, along with GR0.4A/B/C) and higher (GR0.6A/B/C and GR1.0A/B/C) membrane interaction, cellular uptake and intracellular fate of amounts of stabilizer. According to the literature, the presence the nanomaterials represent the most common route for of serum can mitigate the cytotoxicity effect of nanomaterials initiation of cytotoxicity at the cellular level.41 by lowering the direct contact between the materials and cell Closer inspection of BEAS-2B cells in all material-treated membrane. For example, Vranic et al. have found that for both conditions showed the presence of intracellular vacuoles, large (5−15 μm) and small (50−200 nm) size GO, a greater mostly found with residing material inside (see Figure 8 and 12632 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article Figure 8. Uptake profile of GR0.3A/C (25 μg/mL), GR0.6A/C (10 μg/mL), and GR1.0A/C (10 μg/mL) in BEAS-2B cells, assessed by confocal imaging (middle section of z-stacks shown). The top, middle, and bottom panels showed the CMFDA, bright field, and merged channel, respectively. See Supporting Information, Figure S10 for images at higher magnifications. Green = CMFDA dye labeled cells, black = graphene flakes. the Supporting Information, Figure S10). This feature is not of the particle surface amines results in excessive proton pump observed in our previous study on the uptake of different activity and osmotic swelling.42 Other cationic nanoparticles, pyrene derivative-stabilized graphene in BEAS-2B cells, and such as cationic polyamidoamine dendrimers, have also been those materials showed exceptional biocompatibility after shown to induce LMP as well as loss of mitochondrial internalization by the cells.15 membrane potential and apoptosis, with a similar proton It has been widely accepted that positively charged sponge mechanism proposed.43 Furthermore, comparable nanoparticles are efficiently taken up by the cells. However, features of autophagosome-like vacuoles are shown for they were also found to subsequently cause lysosomal BEAS-2B cells treated with single-walled carbon nanotubes membrane permeabilization (LMP), leading to cell death. In that triggered autophagic cell death.44 Autophagy is a the case of the cationic polystyrene nanosphere, for instance, a fundamental catabolic process essential to cell hemostasis “proton sponge” theory has been proposed to explain maintenance; it can be triggered by physical stress and lysosomal rupture, in which the high proton buffering capacity regulates the degradation of damaged organelles.45 The uptake 12633 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article of BPS-stabilized graphene nanosheets and the presence of dense vacuoles hence provide some tentative ideas on the ■ AUTHOR INFORMATION Corresponding Authors cytotoxic mechanism induced by the material. However, Sandra Vranic − Nanomedicine Lab, Faculty of Biology, further work is needed to elucidate the mechanism of Medicine and Health, AV Hill Building, University of cytotoxicity stimulated by the BPS-stabilized graphene nano- Manchester, Manchester M13 9PL, UK; National Graphene sheets. Future research should investigate whether the Institute, University of Manchester, Manchester M13 9PL, materials have triggered lysosomal rupture or autophagy. UK; orcid.org/0000-0002-6653-7156; Overall, a general size-dependent cytotoxicity effect of BPS- Email: sandra.vranic@manchester.ac.uk stabilized graphene was found at lower stabilizer concen- Cinzia Casiraghi − Department of Chemistry, University of trations. As shown by optical imaging and the PI/AV assay, the Manchester, Manchester M13 9PL, UK; orcid.org/0000- smaller graphene nanosheets appear to be more toxic than the 0001-7185-0377; Email: cinzia.casiraghi@ larger ones. The size of the nanomaterial is a well-acknowl- manchester.ac.uk edged factor that plays a critical role in its resultant toxicity effect. However, contradictory observation as to whether Authors smaller or larger graphene-based nanomaterials are more toxic Chen-Xia Hu − Department of Chemistry, University of is seen in the literature,46 which is expected due to the different Manchester, Manchester M13 9PL, UK preparation routes and use of different stabilizers. In the case of Oliver Read − Department of Chemistry, University of our defect-free graphene, the initial amount of the stabilizer is Manchester, Manchester M13 9PL, UK; orcid.org/0000- found to be the more dominant factor in affecting the 0001-9771-6096 cytotoxicity of the material than its size. This is caused by the Yuyoung Shin − Department of Chemistry, University of accumulation of material in intracellular vacuoles, causing Manchester, Manchester M13 9PL, UK lysosomal rupture. As one would expect the amount of Yingxian Chen − Nanomedicine Lab, Faculty of Biology, adsorbed BPS to increase with the size of the flakes, our results Medicine and Health, AV Hill Building, University of show that the cytotoxicity should not only be related on how Manchester, Manchester M13 9PL, UK; National Graphene much stabilizer is adsorbed but also on how the BPS molecules Institute, University of Manchester, Manchester M13 9PL, are adsorbed on the flake as their molecular arrangement can UK change with increasing stabilizer concentration.14 Jingjing Wang − Department of Chemistry, University of Manchester, Manchester M13 9PL, UK 4. CONCLUSIONS Matthew Boyes − Department of Chemistry, University of We demonstrated successful fractioning by LCC of defect-free Manchester, Manchester M13 9PL, UK graphene dispersions in water produced by using the BPS Niting Zeng − Department of Chemistry, University of stabilizer. The cytotoxicity profile of the fractions was Manchester, Manchester M13 9PL, UK investigated, showing that the initial amount of the stabilizer Adyasha Panigrahi − Department of Chemistry, University of is crucial in determining the critical dose at which cellular Manchester, Manchester M13 9PL, UK stress is observed. In particular, for a stabilizer concentration Kostas Kostarelos − Nanomedicine Lab, Faculty of Biology, above 0.4 mg/mL, the cytotoxicity does not depend on lateral Medicine and Health, AV Hill Building, University of size or thickness as all fractions show the same critical dose at Manchester, Manchester M13 9PL, UK; National Graphene which cellular stress is observed. As the stabilizer is not soluble Institute, University of Manchester, Manchester M13 9PL, in water, this suggests that the cytotoxicity is caused by an UK; Catalan Institute of Nanoscience and Nanotechnology excess of molecules adsorbed on the nanosheets. As the (ICN2),, UAB Campus Bellaterra, Barcelona 08193, Spain; cytotoxicity profile changes with the initial amount of orcid.org/0000-0002-2224-6672 stabilizer, this also suggests a different molecular arrangement Igor Larrosa − Department of Chemistry, University of on the nanosheet above a critical stabilizer concentration. We Manchester, Manchester M13 9PL, UK; orcid.org/0000- also observe a strong decrease in the concentration of 0002-5391-7424 dispersed graphene and a reduction in zeta potential when Complete contact information is available at: using a large amount of stabilizer, further confirming a different https://pubs.acs.org/10.1021/acsanm.2c02403 molecular arrangement of the excess BPS molecules, which is somehow detrimental to material exfoliation and colloidal Author Contributions + stabilization as well as to the cytotoxicity. In contrast, when the C.-X.H. and O.R. shared first co-authorship stabilizer concentration is below 0.4 mg/mL, the critical dose Notes at which cellular stress is observed depends on the average size The authors declare no competing financial interest. of the nanosheet, with the largest nanosheets (average lateral size >200 nm) showing a higher critical dose in comparison to the smaller nanosheets. ■ ACKNOWLEDGMENTS This work has been supported by the EPSRC in the framework ■ ASSOCIATED CONTENT * Supporting Information sı of the 2D-Health Programme Grant (EP/P00119X/1). O.R. acknowledges financial support from the Lloyd’s Register Foundation. J.W. acknowledges financial support from the The Supporting Information is available free of charge at National Physical Lab in London. Y.C. acknowledges the https://pubs.acs.org/doi/10.1021/acsanm.2c02403. studentship from the UK Research and Innovation (UKRI) Engineering and Physical Sciences Research Council (EPSRC) TGA profiles, representative AFM images, tabulated Centre for Doctoral Training programme (Graphene NOW- AFM and Raman data, and uptake profiles (PDF) NANO CDT; EP/L01548X/1). The University of Manchester 12634 https://doi.org/10.1021/acsanm.2c02403 ACS Appl. Nano Mater. 2022, 5, 12626−12636 ACS Applied Nano Materials www.acsanm.org Article Bioimaging microscope used in this study was purchased with Vranic, S.; Larrosa, I.; Casiraghi, C. Enhanced Liquid Phase grants from the Biotechnology and Biological Sciences Exfoliation of Graphene in Water Using an Insoluble Bis-Pyrene Research Council (BBSRC), Wellcome Trust, and the Stabiliser. Faraday Discuss. 2021, 227, 46−60. University of Manchester Strategic Fund. The authors (15) Shin, Y.; Vranic, S.; Just-Baringo, X.; Gali, S. 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