Nanoscale This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online PAPER View Journal | View Issue Stable, concentrated, biocompatible, and Cite this: Nanoscale, 2020, 12, 12383 defect-free graphene dispersions with positive Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. charge† Yuyoung Shin, ‡a Sandra Vranic,‡b,c Xavier Just-Baringo, ‡a Sai Manoj Gali,d Thomas Kisby,b,c Yingxian Chen,b,c Alexandra Gkoutzidou,e Eric Prestat, e,f David Beljonne, d Igor Larrosa, *a Kostas Kostarelos *b,d and Cinzia Casiraghi *a The outstanding properties of graphene offer high potential for biomedical applications. In this frame- work, positively charged nanomaterials show better interactions with the biological environment, hence there is strong interest in the production of positively charged graphene nanosheets. Currently, pro- duction of cationic graphene is either time consuming or producing dispersions with poor stability, which strongly limit their use in the biomedical field. In this study, we made a family of new cationic pyrenes, and have used them to successfully produce water-based, highly concentrated, stable, and defect-free graphene dispersions with positive charge. The use of different pyrene derivatives as well as molecular Received 4th April 2020, dynamics simulations allowed us to get insights on the nanoscale interactions required to achieve Accepted 24th May 2020 efficient exfoliation and stabilisation. The cationic graphene dispersions show outstanding biocompatibil- DOI: 10.1039/d0nr02689a ity and cellular uptake as well as exceptional colloidal stability in the biological medium, making this rsc.li/nanoscale material extremely attractive for biomedical applications. 1. Introduction surfactants,10,12–15 polymers16–18 and aromatic molecules,19–24 have been investigated. In particular, pyrene derivatives have Liquid-phase exfoliation (LPE)1 allows for mass-scalable, cost- shown to be more effective exfoliating agents than traditional effective and versatile production of graphene formulations surfactants,21 and have been successfully used to make bio- suitable for a wide range of applications, from composites to compatible and inkjet-printable graphene inks.25 Despite the biological applications.2–5 Organic solvents have been shown success of pyrene derivatives for graphene production, mainly to provide efficient exfoliation.1,6,7 On the other side, LPE in negatively charged or neutral functional groups have been water can also be successfully achieved through covalent studied in detail, leading to production of anionic or neutral functionalisation,8,9 or non-covalent functionalisation with graphene.21–23 Only a few studies have reported exfoliation stabilisers.10,11 Different types of stabilisers, including with cationic pyrene molecules, resulting in poorly concen- trated and/or unstable graphene dispersions, hindering their use in practical applications.21,26,27 Amphoteric pyrene mole- a Department of Chemistry, University of Manchester, Oxford Road, Manchester, UK. cules were also recently used to produce graphene disper- E-mail: cinzia.casiraghi@manchester.ac.uk b Nanomedicine Lab, National Graphene Institute and Faculty of Biology, Medicine & sions,28 but their reduced colloidal stability at neutral pH Health, The University of Manchester, Manchester M13 9PT, United Kingdom. makes this material unsuitable for most applications. One of E-mail: kostas.kostarelos@manchester.ac.uk the major bottlenecks of LPE in water assisted by pyrene c National Graphene Institute, University of Manchester, Booth Street East, derivatives is related to the poor understanding of the nano- Manchester, UK d scale mechanisms giving rise to exfoliation and stabilisation, Catalan Institute of Nanoscience and Nanotechnology (ICN2), UAB Campus Bellaterra, Barcelona, Spain i.e. we still do not know how to design the pyrene derivative e Department of Materials, University of Manchester, Oxford Road, Manchester, UK that can provide the highest exfoliation efficiency. f SuperSTEM Laboratory, SciTech Daresbury Campus, Daresbury, UK In this work, we have designed a new family of cationic † Electronic supplementary information (ESI) available: Synthesis and character- pyrene derivatives that was used to systematically investigate isation of pyrene derivatives; MD simulation results; dispersion stability; AFM the exfoliation efficiency and quality of the material produced analysis; Raman analysis; TEM images; biocompatibility test. See DOI: 10.1039/ d0nr02689a as a function of the charge, type of functional group and ‡ Shared first authorship. length of the linker between the pyrene core and the func- This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 12383–12394 | 12383 View Article Online Paper Nanoscale tional group. Our results show that the most efficient pyrene 2.3.2. Zeta-potential measurements. Electrophoretic mobi- derivatives are those with the polar group far from the pyrene lity (μ) was measured using a ZetaSizer Nano ZS (Malvern base as this configuration minimises interference between the Instruments, UK) after dilution of samples with water in the adsorption of pyrene on graphene and the interaction of the folded capillary cells (Malvern Instruments, UK). Default This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. functional group with the water molecules, in agreement instrument settings for water-based system and automatic ana- with Molecular Dynamics (MD) simulations. We demonstrate lysis were used for all measurements, performed at 25 °C and that stable, highly concentrated, defect-free dispersions, con- at the natural pH. The equipment software automatically taining mostly single and few-layers with average lateral size of converted the μ to zeta-potential (ζ) values by using Henry’s about 200 nm, can be obtained using very small amount of equation: μ = 2εζF(κa)/3η where ε is dielectric constant, η is the stabiliser (0.4 mg mL−1). These cationic graphene dispersions solution viscosity and F(κa) is Henry’s function which is Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. also show exceptional biocompatibility, intracellular uptake approximated to the value of 1.5 using the Smoluchowski profile and stability in the biological medium, even with approximation for polar media, valid for dispersed particles of protein serum, making them a very attractive material for any shape including plate-like particles.29 All values for nanomedicine applications. samples are mean ± standard deviation (SD), calculated from triplicate measurements. 2.3.3. Dynamic light scattering (DLS) measurements. 2. Experimental Hydrodynamic size and polydispersity index of graphene flakes were measured using a ZetaSizer Nano ZS (Malvern 2.1. Materials Instruments, UK) after dilution of samples with water in the Natural graphite flakes were provided by Graphexel Ltd. The disposable polystyrene cuvettes (Malvern Instruments, UK). details of the cationic pyrene synthesis routes and all charac- Default instrument settings for water-based system and auto- terisation methods used for identification of the structure and matic analysis were used for all measurements, performed at purity of the compounds are discussed in the ESI (section S7†). 25 °C and at the natural pH. DLS measurements yielded a 1-Pyrenesulfonic acid sodium salt (PS1) (≥97.0%) was pur- mean value for the size (Z-average), which is calculated based chased from Sigma-Aldrich. Silicon oxide wafers with 290 nm on cumulated analysis of the measured intensity.30 It should double side coating were purchased from Inseto Ltd (UK). be noted that the hydrodynamic size measured by DLS is based on assuming hypothetical hard spherical shaped par- 2.2. Preparation of graphene dispersions ticles that diffuse via Brownian motion,30 which is not ideal Graphene dispersions were prepared via LPE in water following for measuring the size of 2-dimensional graphene sheets, but the protocol developed in previous works.22,25,28 In detail, can be used for estimating the size changes in dispersion. All 30 mg of graphite was added to 10 mL of de-ionised (DI) water, values for samples are mean calculated from triplicate previously mixed with 4 mg of the cationic pyrenes. The measurements. mixture was then sonicated at 300 W for 7 days using a 2.3.4. TEM. Transmission Electron Microscopy (TEM) Hilsonic bath sonicator. Afterwards, un-exfoliated graphite was imaging and Selected Area Electron diffraction (SAED) were removed by 2-steps centrifugation (Sigma 1-14k refrigerated performed using a Philips CM20 and a FEI Talos 200X with centrifuge) at 3500 rpm (903g) for 20 minutes. After each both microscopes operating at an acceleration voltage of 200 centrifugation step, the supernatant containing graphene and kV. For the measurement of the number of layers, High the pyrene derivatives in water was collected. Graphene dis- Resolution TEM (HRTEM) images were acquired on the FEI persions with PS1 were prepared using the same protocol for Talos using a FEI Ceta CMOS camera. TEM samples were pre- direct comparison. Graphene dispersions used for biological pared by drop-casting the graphene dispersions on a lacey study are prepared in larger volume (100 mL) using a 600 W carbon copper grid. Hilsonic bath sonicator. The excess pyrene molecules are 2.3.5. AFM. A Bruker Atomic Force Microscope (MultiMode removed by 2-steps centrifugation (Sigma 1-14k refrigerated 8) in Peak Force Tapping mode, equipped with ScanAsyst-Air centrifuge) at 15 000 rpm (16 600g) for 60 minutes. After each tips, was used to determine the lateral size distribution of the centrifugation step, the supernatant was removed and the sedi- flakes. The sample was prepared by drop casting the solution ment was re-dispersed in DI water. on a clean silicon substrate; several areas of 100 μm2 were scanned and about 200 flakes were selected for lateral size ana- 2.3. Characterisation lysis. Lateral dimension and thickness distributions of gra- 2.3.1. UV-Vis spectroscopy. The final concentration of gra- phene nanosheets were carried out using Gwyddion scanning phene dispersed in the solution was determined using UV-Vis probe microscopy data processing software. spectroscopy. The UV-Vis spectrum of graphene appears flat 2.3.6. Raman spectroscopy. Raman measurements were and featureless in the visible-IR region,1 so the absorption was performed using a Renishaw Invia Raman spectrometer measured at 660 nm. The Beer–Lambert law was used to derive equipped with 514.5 nm excitation line and 2.0 mW laser the concentration by assuming an absorption coefficient of power. Graphene dispersions were dropcast onto silicon sub- 2460 L g−1 m−1 at 660 nm.7,10,12 A PerkinElmer l-900 UV-Vis- strates and measurements were performed on isolated and NIR spectrophotometer was used to acquire the spectra. individual flakes. The Raman spectra were taken with a 100× 12384 | Nanoscale, 2020, 12, 12383–12394 This journal is © The Royal Society of Chemistry 2020 View Article Online Nanoscale Paper NA0.85 objective lens and 2400 grooves per mm grating. CCL-2™, ATCC, LGC standards, UK) were maintained in RPMI Typically, 30–50 flakes were measured for each sample. The 1640 and DMEM high glucose cell culture medium (Sigma- Raman peaks were fitted with a Lorentzian lineshape. Aldrich, Merck Sigma, UK) respectively, supplemented with The Raman analysis was performed using a qualitative pro- 10% FBS (Gibco, Thermo Fisher Scientific, UK), 1000 units This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. tocol for analysing LPE graphene, which has been described penicillin, and 1 mg mL−1 streptomycin (Sigma-Aldrich, Merck previously.31–35 In detail, the shape of the 2D peak was used Sigma, UK) at 37 °C in a humidified 5% CO2 incubator. Cells for determination of the thickness distribution. The 2D peak were passaged twice a week using a 0.05% Trypsin–EDTA solu- was fitted with a single Lorentzian lineshape, which was used tion (Sigma-Aldrich, Merck Sigma, UK) when reaching 80% to distinguish between single-layer graphene (SLG), few-layer confluence. Activity of trypsin was stopped using 10% FBS. All sheets (FLG) and graphitic material (>10 layers with AB stack- experiments were done using cells with a passage number ing) by evaluating the fit residual, R2. SLG showed a single Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. between 25 and 35 (Beas-2B) and 10 and 20 (HeLa). symmetric peak with R2 > 0.987, FLG a single asymmetric peak 2.5.2. Cell treatments. Depending on the experiment, cells with R2 < 0.987, and graphitic material was distinguished by were seeded in 12-well plates (for optical microscopy and PI/ its characteristic peak shape with shoulder. annexin V staining; provided from Corning, Costar, Sigma- Aldrich, Merck Sigma, UK) or in Cellview™ cell culture dishes 2.4. Molecular dynamics simulations (for confocal microscopy; provided from Greiner Bio-One Ltd, The adsorption process of pyrene derivatives on the surface of UK) and treated when reaching 60–80% confluence. graphene layer, in the presence of explicit water molecules, Treatments were performed after dispersing Gr in RPMI 1640 was simulated using MD simulations, performed with the cell culture medium in the presence of 10% FBS at indicated NAMD36 software. All pyrene derivatives were described with a concentrations. Incubations with Gr were maintained for 24 h. customised general AMBER force field37 where in the atomic This experimental design was selected in order to minimize charges and the torsional potentials were calculated at the the effect of different cell culture media composition on aggre- M02/6-311G(d) level of theory and used in the force field fol- gation/agglomeration state of Gr to be able to make compari- lowing the scheme described by ref. 38. Water molecules were son of the effect of graphene flakes across two cell lines represented by the well-known and reliable “modified TIP3P” (BEAS-2B and HeLa). force field.39 A simulation box of size 63.90 × 51.65 × 68.00 Å3, 2.5.3. Confocal microscopy. Cells were seeded in containing an infinite sheet of SLG at the bottom of the simu- Cellview™ cell culture dish (627870, Greiner Bio-One Ltd, UK) lation box, was used for each pyrene derivative. Each derivative and treated when reaching 60–80% confluence with 50 μg was positioned at a perpendicular distance of 2.5 Å from the mL−1 Gr for 24 h. After the treatment, supernatant containing plane of the SLG and the box was filled with 9000 water mole- Gr was removed and cells were stained with Fluorescein cules. In the first step, MD equilibration was performed for 20 Diacetate (FDA, Thermo Fisher Scientific, UK) live cell dye (100 ns under constant pressure (1 bar) and temperature (298 K) nM, 0.5 mL RPMI cell culture medium containing 10% FBS) with the NPT ensemble, and the simulations were extended for shortly before imaging. Cells were examined under a Zeiss 780 an additional 40 ns of production cycle in the NPT ensemble. confocal laser scanning microscope using a 40× objective with Subsequently, free energy of interaction of each pyrene deriva- a confocal mode. Excitation wavelength for the FDA dye was tive with SLG was evaluated by computing the potential of 488 nm, emission maximum was 520 nm. Images were pro- mean force (PMF) of the adsorption process employing the cessed using Zeiss microscope software ZEN. The experiment Adaptive Biasing Force (ABF) method.40 Three different PMF was repeated three times. profiles were calculated by performing three different/indepen- 2.5.4. Flow cytometry. For the PI/annexin V staining experi- dent ABF simulations for 75 ns in the NPT ensemble by taking ment, cells were seeded and treated in 12-well plates after the (i) total center of mass of the pyrene derivative (COM), (ii) reaching 60–80% confluence. After 24 h of treatment, super- isolated center of mass of the pyrene unit of the molecule natants were removed and cells were gently washed once with (PYR) and (iii) isolated center of mass of the functional group phosphate-buffered saline (PBS) containing Ca2+/Mg2+ (Sigma- of the molecule (FUN). For all the ABF simulations, the lower Aldrich, Merck Sigma, UK). Annexin V staining was performed and upper bounds were set to 2 Å and 20 Å, respectively and according to the instructions of the manufacturer (Molecular no specific constraints were employed on the internal degrees Probes, Thermo Fisher Scientific, UK). In brief, cells were tryp- of freedom for all the derivatives, meaning that the derivatives sinised for 5 min, centrifuged at 1500 rpm for 5 min, then re- are free to rotate along the center of mass and to translate suspended in 50 μL of annexin binding buffer (Molecular along the ‘xy’ plane ( plane parallel to the graphene layer). The Probes, Thermo Fisher Scientific, UK), and stained with 1 μL three independent PMF profiles were independently adjusted of annexin V–Alexa Fluor488 conjugate for 20 min at 25 °C. with respect to the free energy at the upper boundary of 20 Å. Propidium iodide (1 mg mL−1, Sigma-Aldrich, Merck Sigma, UK) was added shortly before the analysis to the final concen- 2.5. Biological studies tration of 1.5 μg mL−1. A total of 10 000 cells were analysed on 2.5.1. Cell culture. Human epithelial bronchial immorta- a BD FACSVerse™ flow cytometer using 488 nm excitation and lised cells (Beas-2B, CRL-9609™, ATCC, LGC standards, UK) 515 and 615 nm band-pass filters for annexin V and PI detec- and human epithelial cervical adenocarcinoma cells (HeLa, tion, respectively. The experiment was repeated three times. This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 12383–12394 | 12385 View Article Online Paper Nanoscale 2.5.5. Stability in complete cell culture medium. Four clarity, the pyrene derivatives produced are indicated with the types of graphene dispersions (50 μg mL−1) were dispersed in acronyms: TMAn, IMIn, and PRDn. RPMI 1640 cell culture medium supplemented with 10% FBS In a general synthetic approach, 1-pyrenyl alcohols II were (complete cell culture medium) for 24 h at 37 °C in a humidi- obtained from a supplier or were prepared by reduction of the This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. fied 5% CO2 incubator. After 24 h, graphene was centrifuged at parent carboxylic acid I (Fig. 1C). With all alcohols II in hand, room temperature, for 30 min at 13 000 rpm. The supernatants the corresponding bromides III were synthesised to serve as were discarded, and the graphene-containing pellets were electrophiles in the condensation with trimethylamine or an resuspended in Milli-Q water. This washing step was repeated N-heterocycle (i.e. 1-methylimidazole or pyridine). The present once in order to remove excess unattached biomolecules and strategy gave access to the desired pyrene cations in good electrolytes, enabling the characterisation of graphene by DLS overall yields and allowed access to gram amounts of products. Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. and zeta potential measurements. The experiment was Only in the case of TMA2 an alternative approach was required repeated two times. due to the competing elimination reaction leading to large amounts of 1-vinylpyrene being formed. In this case, 1-pyreny- lacetic acid 1 was coupled with dimethylammonium chloride to form dimethylamide 2, which was subsequently reduced to 3. Results and discussion amine 3 and alkylated with methyl iodide to yield iodide salt 4 3.1. Preparation of cationic pyrenes (Fig. 1D). The desired TMA2 bromide salt was obtained after anion exchange to have a consistent family of cationic pyrenes A family of cationic amphiphilic pyrenes was prepared. The with bromide counter ions. molecules are composed of three parts (Fig. 1A): (i) a pyrene The solubility of the molecules is reported in Fig. S1, ESI.† aromatic ring, which allows adsorption to graphene; (ii) a carbon chain linker of variable length (n = 1–4, where n is the number of methylene groups) between the pyrene ring and the 3.2. Liquid-phase exfoliation of graphene: experimental and polar group; (iii) a cationic group. We have investigated the fol- modelling lowing functional groups: a trimethylammonium group (TMA The obtained graphene dispersions for each cationic pyrene family), as example of tetraalkylammonium substitution; a molecules are shown in Fig. 2A. To better illustrate the concen- 3-methylimidazolium ring (IMI family) to introduce an elec- tration difference, the dispersions were diluted by a factor 10. tron-rich positively-charged aromatic; and a pyridinium ring Fig. 2B shows the graphene concentrations obtained with the (PRD family) to showcase the effect of an electron-poor aro- different cationic pyrene molecules. For comparison, the plot matic cation. The complete family is shown in Fig. 1B. For also reports graphene concentration obtained with 1-pyrene- Fig. 1 (A) Design of cationic pyrenes. (B) Family of cationic pyrenes designed for this study. (C) General synthetic strategy for the synthesis of cat- ionic pyrenes (NC = nitrogen-based cations). (D) Alternative route towards TMA2 synthesis. 12386 | Nanoscale, 2020, 12, 12383–12394 This journal is © The Royal Society of Chemistry 2020 View Article Online Nanoscale Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. Fig. 2 (A) Photographs of graphene dispersions prepared with the cationic pyrenes (diluted by ×10), and (B) evolution of the final graphene concen- tration with carbon chain length and zeta potential measurements for the graphene dispersions (for PS1, |ζ| is shown). (C) Single layer graphene per- centage determined by Raman spectroscopy and mean flake size estimated from AFM measurement, (D) lateral size and number of layers counted from TEM measurement for three samples, TMA4, IMI1 and PRD1. sulfonic acid sodium salt (PS1), which is one of the most used effective at lowering surface tension for increasing chain anionic pyrene derivative for LPE.22,23 Fig. 2B shows that dis- length.41 This result can be tentatively explained by taking into persions made with IMI1 and PRD1 have similar or higher con- account that as the charge gets further away from the hydro- centration than those produced with PS1, while the TMA1 phobic core, the interaction between the polar group and the stabiliser is not efficient at exfoliating graphite. However, the water molecules and the π–π interaction between the pyrene concentrations drastically change with increasing chain and the graphene surface are better separated, i.e. they do not length: the concentration of graphene prepared by TMA interfere with each other, by enhancing the adsorption of the strongly increases with increasing n, becoming comparable, if hydrophobic pyrene on the surface of graphene as well as pro- not even higher than the concentration obtained by IMI and viding better hydration of the polar group. When the func- PRD families (Fig. 2B). Note that this behavior cannot be tional group is too close to the pyrene base, as in the case of explained by the different solubility of the molecules, as there TMA1, the bulky functional group interferes with the adsorp- is no apparent correlation between solubility (Fig. S1†) and tion of the aromatic planar surface of the pyrene and graphene graphene concentration (Fig. 2B). The increase in efficiency surface. As the distance between the polar functional group with increasing distance of the polar group from the hydro- and the hydrophobic base increases, steric hindrance is sig- phobic region of the molecules, seen for the TMA family, is nificantly reduced, while the interaction of the localised observed in traditional ionic surfactants, which are more charge on the functional group and the water molecules is This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 12383–12394 | 12387 View Article Online Paper Nanoscale enhanced, therefore improving the stabilisation of graphene (−18.9 kcal mol−1) and PRD3 (−18.7 kcal mol−1) are higher in flakes in aqueous media. On the other hand, in case of IMI value compared to that of PRD1 (−19.3 kcal mol−1) and PRD4 and PRD families, a more complex pattern is observed: rela- (−20.3 kcal mol−1). tively high exfoliation efficiency was achieved at n = 1, followed A similar trend is also observed for the PMF profiles of the This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. by a dramatic decrease at n = 2, and then a slow increase of pyrene base (Fig. 3B, middle panel). These observations indi- exfoliation was observed with increasing n. For n = 1, the aro- cate stronger interaction of PRD with graphene for n = 1 and 4, matic functional groups as well as the pyrene base show strong driven by the interaction of both the functional group and the interaction with the graphene surface, possibly facilitating the pyrene base with the graphene surface. This explains the adsorption of the stabilisers on the graphene surface, hence different exfoliation trends observed with increasing n for the high exfoliation efficiency. TMA: the interaction with the graphene surface is driven Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. Note that linear alkyl chains are known to form stable mainly by the pyrene base for TMA family and the TMA func- layers on graphene.31,33 However, the length of the chain in tional group does not show any variation in the free energy our pyrene derivatives is very short, hence this effect is unlikely minimum but prefers to interact with water molecules, in com- to be responsible for the higher exfoliation efficiency. Indeed, parison to the PRD functional group. This has been confirmed PRD1 and PRD4 shows similar graphene concentration. by visualising the relative orientation of TMA and PRD deriva- Getting insight into the molecular interactions during exfolia- tives on the graphene layer. The MD snapshots, taken from the tion is of fundamental importance because it enables design production run of 40 ns in NPT ensemble with the atomic of the pyrene derivatives providing the highest exfoliation positions of the pyrene derivatives averaged over 20 equally efficiency. Thus, the adsorption process of each cationic separated time frames, are shown in Fig. 3C. This figure shows pyrene molecule on graphene surface in aqueous medium was that the interaction of the TMA functional group with the sur- assessed by employing MD simulations.23 Each cationic pyrene rounding water molecules tends to improve with increasing n, was placed in a box filled with 9000 water molecules at a start- due to the increase in the relative stability of the localised posi- ing distance of 2.5 Å from the basis plane consisting of a tive charge on the nitrogen interacting with water molecules. single layer graphene sheet. The potential of mean force (PMF) For PRD, on the other hand, both the pyrene base and the of the adsorption process was calculated using adaptive functional group of the molecules are strongly interacting with biasing force (ABF) simulations (see Experimental section for the graphene layer. As shown in Fig. 3C (bottom), for PRD1 more details). To assess the different factors influencing the and PRD4, the plane of the pyrene base and the aromatic func- interaction of graphene with cationic pyrene molecules in the tional group are both parallel to the surface of graphene, presence of water, three independent PMF profiles were whereas for PRD2 and PRD3, the functional group is energeti- obtained by performing different ABF simulations considering: cally frustrated, leading to a decreased interaction with the gra- (1) the center of mass of the whole pyrene molecules (COM), phene layer. This is further illustrated by the calculation of the (2) the pyrene base (PYR) and (3) the functional group (FUN). tilt angle between the vector perpendicular to the pyrene base Fig. 3A shows the PMF curves obtained in the three cases and the vector perpendicular to the graphene surface for the TMAn family. The largest changes observed with (Table S1†): the calculated tilt angles of PRD2 and PRD3 pyrene increasing n are seen for the functional group (right panel): are much lower (166° and 154°, respectively) than that of PRD1 with the increase in n, the PMF profile changes from exhibit- and PRD4 pyrenes (∼180°). Similar energetic profiles are ing a global minimum (located at the same energy, indepen- expected for IMI family, as the IMI functional group is similar dent of n) to that of exhibiting an additional broadened local to that of PRD in terms of energy delocalisation and hence, minimum, allowing the charged functional group to be farther the same mechanism is expected to govern the energetic inter- away from the graphene surface and to freely interact with actions between graphene and IMI derivatives. water molecules. Similarly, the PMF curves of the pyrene core Our results show that higher exfoliation efficiency is show one local minimum at ∼3.5 Å, independent from n, as obtained with pyrene derivatives with long chain length (n > shown in Fig. 3A, middle. 3). In case of short chain length, the exfoliation efficiency will However, when n = 1, the energy minimum is much higher strongly depend on the type of functional groups used, with (−16.9 kcal mol−1) compared to that observed with n > 1 IMI or PRD outperforming TMA due to the increased affinity (−18.2 kcal mol−1), indicating a better interaction of the to graphene. pyrene base with the graphene surface when n > 1. Fig. 3A, left panel shows the combined effect of pyrene adsorption 3.3. Characterisation of the exfoliated graphene nanosheets (Fig. 3A, middle) and hydration of the functional group Fig. 2B (bottom) shows that all the graphene dispersions pre- (Fig. 3A, right), with increasing stabilisation energy for increas- pared in this study have a relatively high zeta potential of ing n, in agreement with the experimental results. ∼40 mV. The zeta potential is typically used as an indicator of In the case of the PRDn family, the PMF profiles of the func- the stability of colloidal suspension: a zeta potential modulus tional group show a variation in the free energy minima, higher than 30 mV is associated to a stable suspension in depending on n (Fig. 3B, right): the global minimum of PRD1 aqueous media.14 To further confirm the stability of the dis- and PRD4 is lower in energy than that of PRD2 and PRD3. As persions, the material was monitored for at least 10 months: shown in Fig. 3B (left panel), the free energy minima for PRD2 no notable sedimentation was observed and no change in the 12388 | Nanoscale, 2020, 12, 12383–12394 This journal is © The Royal Society of Chemistry 2020 View Article Online Nanoscale Paper This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. Fig. 3 PMF profiles of pyrene derivatives with (A) TMA and (B) PRD functional groups for (top) the center of mass, (middle) pyrene base and (bottom) the functional group. (C) MD snapshots of pyrene derivatives with (left) TMA and (right) PRD functional groups. zeta potential measured after 10 months’ storage was reported as further broadening of the 2D peak is observed as result of (Fig. S4†). This result shows that the dispersions are stable, the nanosheets edges, adsorption of solvent and stabiliser regardless of which cationic pyrene is used for exfoliation. molecules, and random re-stacking of the flakes.22,45 We per- This is in contrast with the concentration, which strongly formed qualitative thickness analysis by using a method pre- depends on the type of pyrene derivative used. viously tested in our group (Methods).22,31,33–35 This is based Raman spectroscopy is one of the most popular techniques on the fit of the 2D peak with a Lorentzian lineshape. Fig. 2C for characterisation of graphene.42,43 Fig. S8† shows represen- and Table S2† show that all twelve dispersions contain more tative spectra taken on ∼40 individual flakes drop-cast on than 20% single layer graphene, which matches rather well silicon substrate for each dispersions. The Raman spectrum with the typical value observed in graphene dispersions shows the characteristic G and D peak at ∼1580 cm−1 and obtained with PS1 (∼30%).22 Notably, the highest single layer ∼1350 cm−1, respectively.43 The D peak is characteristic of gra- graphene percentage (>40%) is observed by using IMI4 and phene produced by LPE2 and it is activated by the edges of the TMA4, suggesting that longer chains may help in achieving not nanosheets,44 having smaller size than that of the laser spot. only higher yield, but also better exfoliation, when used in The 2D peak at 2680 cm−1 is also observed in the high energy combination with specific functional groups. In contrast, the region of the spectrum. Note that this peak is typically used to concentration of single layer graphene obtained by the PRD identify single-layer graphene, having a full width at half family seems to be less sensitive to the chain length. maximum (FWHM) of around 30 cm−1.43 However, this identi- We further investigated the graphene flakes by atomic force fication method does not work for graphene produced by LPE microscopy (AFM) and transmission electron microscopy This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 12383–12394 | 12389 View Article Online Paper Nanoscale (TEM). Fig. 2C shows the mean flake size measured by AFM. applications is based on the use of functionalisation of gra- The thickness and size distribution histogram for all the phene oxide (GO) and reduced GO (rGO) with cationic poly- samples can be found in ESI (Fig. S5 and S6†). Note that the mers such as polyethylenimine (PEI) and polyethylene glycol theoretical thickness of single layer graphene is estimated to (PEG). However, production of GO involves use of acid and This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. be ∼0.34 nm, but because of surface adsorption of stabilisers washing steps,2 and the intrinsic negative surface charge of and/or solvent molecules, AFM often shows higher thickness: GO requires complex multiple steps for such functionalisation. typically, a thickness of 1–2 nm for single layer graphene pro- Furthermore, its oxygen-containing functional groups may duced by LPE has been reported.22,46,47 Thus, AFM cannot be lead to oxidative stress in biological environment,53–56 for used to derive quantitative information on the thickness. The example it has been reported that PEI-functionalised GO AFM analysis shows that the lateral size distribution of the gra- shows cytotoxicity and reduced colloidal stability.57–59 Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. phene nanosheets is between 50 nm and 400 nm, with average Particularly, reduced colloidal stability of GO or functionalised lateral size of ∼200 nm, for all the graphene dispersions pre- GO in biological medium, especially in presence of protein pared in this paper (Fig. 2C). serum, can lead to different cytotoxicity and cellular uptake Three dispersions, obtained with TMA4, IMI1 and PRD1, due to the agglomeration by biocoronation.57–60 Hence, were selected for TEM measurement, as they are characterised alternative oxygen-free graphene-based materials are extremely by the highest graphene concentration values for each cationic attractive for biomedical studies. group-type. Distribution histograms of lateral size and number For the biomedical study, we selected graphene dispersions of layers of those dispersions are shown in Fig. 2D. The lateral with the highest concentration of graphene flakes, namely size distribution is similar to that one measured by AFM those produced with TMA3, IMI1 and PRD1. Note that instead (Fig. S5†). Compared to the other two dispersions, the dis- of TMA4, TMA3 was used since it gives similarly high concen- persion prepared with TMA4 showed a higher percentage of tration of graphene flakes with easier synthesis of the stabil- smaller flakes (<300 nm), but still within the expected range iser. Graphene dispersions obtained using PS1 25 were also for LPE graphene dispersions. The number of layers of gra- added for comparison. Note that the thickness and size distri- phene nanosheets was counted from individual flakes in the bution are similar in all dispersions, i.e. average size of High Resolution TEM image. These measurements indicate ∼200 nm and thickness of less than 7 layers for most of the that the dispersions are mostly composed by few layers gra- flakes. Note that other graphene-based materials with similar phene nanosheets (<7 layers), where the sample prepared with thickness and size have been shown to be able to penetrate the TMA4 shows the higher percentage of thinner layers, in accord- membrane, but it is unclear if and how the surface chemistry ance with the Raman analysis (Fig. 2C). The disagreement in and charge may impact on the intracellular uptake. The the single layer graphene percentage between Raman and TEM samples are labelled as follows: Gr-TMA3, Gr-IMI1, Gr-PRD1 measurements is attributed to the difference in resolution and Gr-PS1. The first three samples contain graphene flakes between the two techniques and in the different sample prep- with positive surface charge (Gr+), while the last one contains arations. In any case, both techniques indicate that the disper- graphene with negative surface charge (Gr−). sions mostly contain thin layers (<7 layers). These results show that the thickness and size distribution of the nanosheets is 4.1. Stability of graphene in cell culture medium similar amongst the three graphene dispersions obtained with Let us first focus on stability of the graphene dispersions in the most efficient cationic pyrene stabilisers. biological solutions, such as cell culture medium, as this can govern both cellular interactions and subsequent responses.61–63 Furthermore, the aggregation of nanomaterials 4. Biological studies in biological media is often associated with data misinterpre- tation and artifacts,64,65 making characterisation of nano- It is well known that dimensionality, lateral size, charge, materials in biologically relevant media as a prerequisite for surface functionalisation and chemical composition of the accurate interpretation of the results. Excellent colloidal stabi- nanomaterial can lead to radically different interactions with lity of nanomaterials is crucial for successful biomedical appli- living systems.48–50 In particular, cationic nanomaterials are cations and clinical translation. typically preferred for biomedical applications because, due to The stability of the graphene dispersions was therefore their positive charge, they show better interactions with the assessed after incubation in complete RPMI1640 cell culture biological environment, such as better cellular internalisation medium using DLS and surface charge measurements due to stronger affinity to the negatively charged biomaterials (Fig. S11 and Table S3†). Although DLS characterisation is such as small interfering ribonucleic acid (siRNA).51 However, valid only for spherical nanoparticles, it can be used to detect the charge is also a significant contributor to the nano- changes in size and polydispersibility of graphene material’s toxicity.52 Thus, the development of non-toxic cat- dispersions,53,66,67 in combination with other characterisation ionic nanomaterials is of fundamental importance in techniques. Table S3† shows that all graphene dispersions nanomedicine. have monodisperse size distribution upon incubation in com- In the framework of 2-dimensional materials, the most plete cell culture medium for 24 h. This is confirmed by the used method to produce cationic graphene for biomedical lack of sedimentation/precipitation of material upon incu- 12390 | Nanoscale, 2020, 12, 12383–12394 This journal is © The Royal Society of Chemistry 2020 View Article Online Nanoscale Paper bation in the cell culture medium (Fig. S11,† insets). containing 10% fetal bovine serum (FBS). The healthiness of Interestingly, the overall surface charge of all four types of gra- the untreated or monolayers cells exposed to 50 µg mL−1 of phene after incubation was found to be negative. However, it is the four types of graphene has been compared (Fig. S12A†). No important to note that the zeta potential measurement is done evident morphological changes on the cellular level (e.g. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. upon incubation in complete cell culture medium (in acellular rounding up of the cells, detachment from the support, loss of conditions), followed by centrifugation, washing and finally re- intercellular connections, swelling of the lysosomes) were dispersing in water. As the zeta potential cannot be measured observed, demonstrating that the graphene flakes were not in situ, the sign of the zeta potential measured may not be cytotoxic even at the highest concentration used (50 µg mL−1). indicative of the zeta potential of the material when in contact Cell viability after the treatment with increasing concen- with the cells. Furthermore, the change in surface charge is trations of graphene materials was further quantified using Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. not specific to graphene based materials: the same result has Annexin V/Propidium Iodide assay. The cells appeared been reported for other positively charged nanomaterials and unstained for indicators of apoptotic and/or necrotic cell attributed to their coating with proteins from the serum.68,69 death, confirming that graphene material is not cytotoxic up to the concentration of 100 µg mL−1 (Fig. 4B and Fig. S13B†). 4.2. In vitro uptake and biocompatibility Thus, no matter the charge or the functional groups, graphene The assessment of cytotoxicity and cellular uptake is funda- produced by pyrene derivatives (at least those investigated in mental to establish the safety profile, but also to explore poten- this work) shows exceptional biocompatibility. To the best of tial therapeutic applications (such as delivery of small thera- our knowledge there are not studies on positively charged gra- peutic molecules or gene therapies) of nanomaterials.70 The phene flakes in vitro. cytotoxicity profile has been established using optical Moving to the intracellular uptake, due to the high light microscopy and further quantified using flow cytometry on absorption capacity of graphene, the material can be easily two cell lines: Beas-2B and HeLa (Fig. S12,† Fig. 4B and detected using bright-field imaging. The optical inspection Fig. S13B†). BEAS-2B cell line was selected as a representative already shows that Gr+ interact more with the two cell lines, of healthy lung epithelium, commonly used in nanotoxicology compared to Gr− (Fig. S12A,† bright-field images). For more to assess the safety of nanomaterials.71,72 HeLa cell line was explicit determination of intracellular uptake of the material, chosen as a representative cancer cell line, commonly used in fluorescein diacetate dye (FDA) was used to stain the inside of the studies focusing on applications of nanomaterials (in par- the cells with green fluorescent signal, which will be quenched ticular graphene-based) for drug delivery.73 The two cell lines where graphene is localised intracellularly (Fig. 4A, Fig. S13A were exposed to increasing concentrations of graphene in com- and S14,† black spots in images showing FDA stained cells). plete cell culture medium, i.e. RPMI1640 cell culture medium This approach clearly shows that the uptake occurs for all four Fig. 4 Uptake and cytotoxicity of Gr in BEAS-2B cells. Uptake and cytotoxicity of Gr in BEAS-2B cells. (A) BEAS-2B cells were exposed to 50 µg mL−1 of Gr-PS1, Gr-IMI1, Gr-PRD1 or Gr-TMA3 for 24 h. After the treatment, cells were stained using fluorescein diacetate (FDA) dye and the uptake of the flakes was estimated using confocal microscopy, by exploiting fluorescence quenching of the FDA dye due to the presence of the flakes inside the cells. Staining of the cells: FDA – green. Scale bar is 10 µm. Assessment of the cytotoxicity of Gr by flow cytometry using Propidium Iodide/Annexin V staining for BEAS-2B cells were exposed to 100 µg mL−1 of Gr for 24 h. In the bivariate plots, live cells are represented in the lower left (LL) quadrant, early apoptotic in the lower right (LR), late apoptotic and/or necrotic cells are shown in the upper right (UR) and necrotic cells are in the upper left (UL) quadrant. This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 12383–12394 | 12391 View Article Online Paper Nanoscale types of graphene. However, Gr− is the least internalised by the Conflicts of interest cells, whereas all Gr+ are taken up more efficiently, even though to a different extent, after 24 h of treatment at the used The authors declare no competing financial interest. dose of 50 µg mL−1 (Fig. 4A, Fig. S13A and S14† overlay This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. images), possibly related to the different surface chemistry between the three Gr+. This result confirms that positively Acknowledgements charged nanomaterials are better taken up by the cells due to the higher affinity of interaction with negatively charged pro- This work has been supported by the EPSRC in the framework teoglycans on the plasma membrane of the cells.74–76 of the project 2D Health (EP/P00119X/1) and the M-ERA.NET Our results clearly demonstrate that defect-free and cationic project MODIGLIANI ( project2110). Computer time for this Open Access Article. Published on 25 May 2020. Downloaded on 1/11/2022 10:19:41 AM. graphene flakes produced by pyrene derivatives have excellent study was provided by the Consortium des Equipements de stability in the medium, and exceptional biocompatibility, Calcul Intensif (CECI, http://www.ceci-hpc.be), and particularly after being efficiently internalised by the cells: this is a very those of the Technological Platform on High-Performance remarkable finding as alternative materials with positive Computing, for which we gratefully acknowledge the financial surface charge, such as cationic nanoparticles and polymers, support of the FNRS-FRFC (Conventions No. 2.4.617.07.F have been shown to induce cell death via lysosomal or rupture and 2.5020.11). DB is FNRS Research Director. CC strongly of the plasma membrane.47,74,75 acknowledges very useful discussions with Lucia Delogu. 5. Conclusions References In this study, various cationic pyrene derivatives were designed 1 Y. 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