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One-pot Green Synthesis of Biocompatible Graphene Quantum Dots and their Cell Uptake Studies Arnab Halder, Maria Godoy-Gallardo, Jon Ashley, Xiaotong Feng, Tongchang Zhou, Leticia Hosta-Rigau, and Yi Sun ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00170 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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ACS Applied Bio Materials

One-pot Green Synthesis of Biocompatible Graphene Quantum Dots and their Cell Uptake Studies Arnab Halder, Maria Godoy-Gallardo, Jon Ashley, Xiaotong Feng, Tongchang Zhou, Leticia Hosta-Rigau and Yi Sun* Department of Micro- and Nanotechnology, Technical University of Denmark, DK-2800 Kgs Lyngby, Denmark ABSTRACT: Graphene-based quantum dots (GQDs) are attractive fluorophores due to their excellent photoluminescence properties, water solubility, low cost and low toxicity. However, the lack of simple, efficient and environmental-friendly synthesis methods often limits their biological applications. Herein, we explore a novel one-pot green synthesis approach for the fabrication of fluorescent GQDs without involving any harsh reagents. Graphene oxide is used as a precursor and a two-hour hydrothermal synthesis is carried out with assistance of hydrogen peroxide, and no further post-purification steps are required. The effects of reaction conditions on the characteristics of GQDs are comprehensively investigated. The as-synthesized GQDs show high photostability and excellent biocompatibility as revealed by cell viability assays for three different cell lines, namely macrophages, endothelial cells and a model cancer cell line. The detailed studies of cellular uptake mechanisms suggest that the for all the three cell lines, the major internalization route for GQDs is caveolae-mediated endocytosis followed by clathrinmediated endocytosis at a less extent. Our results demonstrate the great potential of the assynthetized GQDs as fluorescent nanoprobes. The study also provides unique insight into the cell-GQDs interactions, which is highly valuable for bio-imaging and other related applications such as diagnostics and drug delivery.

KEYWORDS:

graphene

quantum

dots,

green

synthesis,

hydrothermal

reaction,

biocompatibility, cellular uptake.

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1. INTRODUCTION Graphene-based quantum dots (GQDs) as a new type of fluorophores have attracted a lot of attention.1,2 Classically, GQDs are small fragments of single or multi-layered π-conjugated 2D graphitic structures with different oxygen-containing functional groups on the edge.3 Due to quantum confinement and the ‘giant red edge effect’, GQDs show excitation wavelengthdependent fluorescence (FL).4 Furthermore, GQDs also have a lot of interesting properties as demonstrated by other members of the graphene family, such as excellent mechanical flexibility, high thermal conductivity, good electron mobility, high 2D surface area, and availability of oxygen-containing functional groups for further functionalization.5,6 Owing to these excellent physicochemical properties, GQDs are particularly interesting for many potential biological applications.7,8 Typically, GQDs synthesis pathways are categorized into two major classes: top-down approach where graphene sheets are cut into flakes, and bottom-up approaches where small aromatic monomers are fused into larger ones.9 Since the bottom-up synthesis of GQDs is often limited by the complexity of the processes and the low-yields, most of the synthesis pathways rely on top-down approaches such as chemical oxidation and exfoliation,10 electrochemical exfoliation,11 hydrothermal/solvothermal treatments3 or microwave/ultrasound assisted methods.12 As graphene-based precursors are mainly inert in nature, the majority of the synthesis processes involve treatment with different strong acids, oxidants and harsh chemicals (e.g., sulphuric acid, nitric acid, potassium permanganate, Sodium borohydride or Potassium dioxide) as ‘chemical scissors’ for cutting down those precursors into smaller sized quantum dots (QDs).13 However, these chemicals usually leave a fair amount of residues with the as-synthesized GQDs, which are


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very difficult to get rid of. Consequently, long post-processing steps (e.g., repeated washing or dialysis for few days) are required to obtain pure GQDs.14 As such, enormous efforts have been devoted to exploring more environmental-friendly chemical scissors.15 Recently, Xing et al. reported that graphene can be degraded by hydrogen peroxide (H2O2).16 Since H2O2 is a powerful oxidant and can avoid the production of toxic by-products during the reaction at high temperature, low concentration of H2O2 can be utilized as an ideal green chemical scissor in top-down synthetic approaches. Several groups have attempted to use H2O2 to synthesize GQDs, nonetheless, these works have been limited by either difficulty in synthesizing small sized GQDs or complicated reaction steps (Table S1). Zhou et al.17 and Jiang et al.18 developed a top-down approach for the synthesis of GQDs by using photo-fenton reagents (H2O2 and FeCl3) and UV irradiation, but the size of the GQDs was as big as 40 nm. Li et al.19 introduced a H2O2-assisted approach to synthesize GQDs by using cyclic voltammetry and UV radiation, whereas the GQDs still needed to be purified by dialysis for 6 days to remove the electrolyte. Jiang et al.20 and Liu et al.21 reported the synthesis of GQDs by using both H2O2 and NH4OH in hydrothermal reactions. The reaction required long time (8-24 h) and extra heating steps to evaporate unreacted reagents. Lately, Lu et al. reported a H2O2–only hydrothermal method to synthesize GQDs from black carbon.24 Although the method eliminated the need of post purification, according to the recommendation of the International Agency for Research on Cancer, carbon black can be carcinogenic to human being (Group 2B).24 Furthermore, compared to graphene-based materials, carbon black has a more irregular structure25 which poses a challenge for the synthesis of uniformly sized GQDs. In contrast, graphene oxide (GO) is a low-cost commercially available material,26 and due to its predefined


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graphene-like structure and pseudo single-sheet nature and reactivity,6 GO can be the ideal material for the synthesis of well-defined GQDs. Herein, we report a simple, fast and one-step green method to synthesize GQDs by using presynthesized GO as a starting material and a low-concentration of H2O2 as an oxidant or ‘chemical scissor’. The effects of different reaction parameters such as the concentration of H2O2 as well as reaction time and temperature are comprehensively investigated. Under optimized conditions, the synthesized GQDs are uniformly small-sized (~5 nm) and photostable. The synthesis process does not involve any major time-consuming post-processing steps. Moreover, as no other toxic chemicals are involved in their synthesis, the GQDs possess excellent biocompatibility. Previously, bio-imaging with GQDs has been reported,27 whereas the toxicity of GQDs and their cellular uptake mechanisms are poorly understood.28,29 In this study, we also evaluate the feasibility of the as-prepared GQDs for bio-imaging applications. We employ three different cell lines, namely macrophages (RAW 264.7), which are the first line of defence of the human body against intruding pathogens; endothelial cells (HUVEC), which are the cells lining our blood vessels; and HeLa cells as model cancer cell line. Cell viability assays are conducted to assess the biocompatibility of the GQDs. Finally, we systematically study the cell internalization pathways that GQDs utilize to penetrate into the different types of cells. 2. EXPERIMENTAL SECTION 2.1. Chemicals and materials characterization: Graphite flakes (< 20 µm, synthetic), hydrogen peroxide (H2O2) concentrated sulphuric acid (H2SO4), concentrated hydrochloric acid (HCl), phosphorus pentoxide (P2O5), potassium persulfate (K2S2O8), potassium permanganate (KMnO4) and quinine sulphate were purchased from Sigma-Aldrich (USA), and were all used as starting materials for preparation of graphene


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oxide (GO). All other chemicals (at least analytical grade) including Dulbecco’s Phosphate Buffered Saline (PBS), Dulbecco´s Modified Eagle´s Medium-high glucose (DMEM D5796), penicillin-streptomycin, sodium pyruvate, fetal bovine serum (FBS), trypsin, PrestoBlue Cell Viability Reagent, paraformaldehyde (PFA), tetramethylrhodamine B isothiocyanate (TRITC)labelled phalloidin (phalloidin-TRITC), adenocarcinoma cell line HeLa, endothelial cell line HUVEC, chlorpromazine hydrochloride, amiloride hydrochloride hydrate, filipin complex from Streptomyces filipinensis, latrunculin A were also purchased from Sigma-Aldrich and used as received. Endothelial Growth Medium-2 Bullet kit (EGM-2) was purchased from Lonza. The EGM-2 is composed of Endothelial Basal Medium (EBM), human epidermal growth factor (hEGF), vascular endothelial growth factor (VEGF), R3-insulin-like growth factor-1 (R3-IGF-1), ascorbic acid, hydrocortisone, FBS, human fibroblast growth factor-beta (hFGF-β), heparin and gentamicin/amphotericin-B (GA). The macrophage cell line RAW 264.7 was obtained from European Collection of Authenticated-Culture Collections (ECACC). X-ray photoelectron spectroscopy (XPS) analysis was performed on a Thermo Scientific™ KAlpha+™ X-ray photoelectron spectrometer system with an Al K-Alpha (1486 eV) X-ray source. All samples were measured by depositing the material on polished Si wafers by drop casting. In all measurements the X-ray spot area was set to 400 µm and a flood gun (to deliver electrons to the sample surface) was used for charge compensation. Raman spectroscopy was conducted on a Thermo Scientific DXR Raman spectrometer equipped with a 455 nm laser. Fourier transform infrared spectra (FTIR) were recorded by a Perkin Elmer Spectrum. X-ray diffraction (XRD) analysis was carried out with a Bruker DB Advance diffractometer unit. FL spectra were recorded using a Tecan Spark® multimode plate reader. UV-VIS spectra were


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recorded using a Shimadzu UV-2600 spectrometer. Transmission electron microscopy (TEM) analyses of samples were carried out on a Tecnai G20 operating. Zeta potential were conducted using Malvern DLS Zetasizer. 2.2. Synthesis of graphene quantum dots (GQDs): For the synthesis of GQDs, 40 mg of pre-synthesized GO precursor was mixed with 30 mL of H2O2 (2-6%) and then each mixture was transferred to a 50 mL Teflon-made autoclave reactor which was subsequently heated to 180 °C for 2 hrs. After cooling down to room temperature, the resultant light yellow solution was filtered using a 0.2 µm paper filter to remove unreacted GO and the resultant GQD filtrate was collected. The as-synthesized GQDs were then freeze dried to obtained a solid samples were stored at room temperature under dark environment for further studies. For all the photoluminescence measurements, the stock GQDs solutions were prepared by dissolving the GQDs Milli-Q water. For pH stability studies, the freeze-dried GQDs were dissolved at three different pH (pH 4, 7 and 10)-based solutions. To measure the stability of the fluorescent signal, the FL intensity of one batch of sample stored under dark conditions was measured at regular intervals.

2.3. Quantum yield (QY) measurement: The QY of GQDs was measured using quinine sulfate (QY = 0.54 in 0.1 M H2SO4) as the standard sample.30 The QY was calculated by using the following equation:

Φ = Φ

(1)


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where Φ refers to the QY, I is the measured emission intensity, A is the absorbance, and η is the refractive index. The subscript “st” represents the standard solution, and “x” represents the sample. 2.4. Cell experiments: Both the mouse macrophage Raw 264.7 and the HeLa cell lines were cultured in DMEM supplemented with 10% (v/v) FBS, 1% sodium pyruvate, 1% penicillin/streptomycin (10 000 U mL-1 and 10 µg mL-1), 2% (v/v) HEPES at 37 °C in a humidified incubator and 5% (v/v) CO2. HUVEC cells were grown in EGM-2 medium supplemented with 2% FBS and endothelial cell growth supplements at 37 °C and 5% (v/v) CO2. For cell viability assays, 30,000 cells (Raw 264.7) and 15 000 cells (HUVEC and HeLa) were seeded in 96-well plates in 200 µL of media. For cell association/uptake experiments and cellular uptake pathway studies, 50 000 cells (Raw 264.7) and 25 000 cells (HUVEC and HeLa) were seeded in a 48-well plate in 500 µL media. In all cell lines, the cell media was renewed every 2 days and cells between passage three and passage five were used for all experiments. Moreover, cells were allowed to attach for 24 h at 37 ˚C and 5% (v/v) CO2. 2.5. Cell Viability Assays: After cell attachment for 24 h, the cells were washed 3× in PBS. Next, 300 µL of GQDs in cell media at different concentrations were added to the wells and incubated for 4 and 24 h, respectively, at 37 °C and 5% (v/v) CO2. After the incubation times, the media of the wells was aspirated carefully and washed 3× in PBS and 90 µL of fresh media with 10 µL of PrestoBlue was added. The cells were incubated for 1 h at 37 °C and 5% (v/v) CO2 and then the solutions were transferred to a different 96-well plate and analysed using a TECAN Spark multimode plate


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reader by measuring the FL of the reduced PrestoBlue at 590 nm when excited at a wavelength of 560 nm. Cells without GQDs were used as high control samples and media only was used as low control sample. The normalized cell viability (nCV) was calculated as % = (experimental value-low control value)/ (high control value-low control value) × 100. Each condition was evaluated by triplicate in three independent experiments. The statistical differences between the different conditions were evaluated using one-way ANOVA with a confidence level of 95% (α = 0.05) using Tukey´s multiple comparison posthoc test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001) in GraphPad Prism 7 software. 2.6. Cell association/uptake experiments: Cells were incubated with a solution of GQDs (1 mg mL-1 for RAW 264.7 and HeLa, and 0.5 mg mL-1 for HUVEC cells) in 200 µL media for 4 and 24 h, respectively, at 37 ˚C and 5% (v/v) CO2. After the incubation times, the cells were washed 3× in PBS in order to remove noninternalized GQDs and were harvested from the wells for flow cytometry analysis. While RAW 264.7 cells were scrapped from the well, HUVEC and HeLa cells were detached from the wells by incubating them in 200 µL trypsin. For FL imagining, the cells were seeded in 48-well plates equipped with a sterile cover glass in each well. After the incubation times, the cells were washed 3× in PBS to remove noninternalised GQDs and fixed with a 4% PFA solution for 30 min at room temperature. Next, the cells were washed 3× in PBS and the actin filaments were stained by incubating them in a solution of phalloidin-TRITC (0.003 mg mL-1) for 1 h at room temperature. Finally, the cells were widely washed in PBS and imaged with an Olympus inverted microscope IX83 equipped with a 100 W mercury lamp for illumination and a 60× objective immersed in oil. Fitted


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appropriate excitation and barrier filters were used to examine cells labelled with phalloidinTRITC and the GQDs.

2.7. Cellular uptake pathway: After the incubation times, the cells were washed 3× in PBS and the RAW 264.7 cells were preincubated with fresh growth medium without penicillin and supplemented with 10 µM chlorpromazine, 1µg mL-1 filipin, 50 µM amiloride or 1 µM lantrunculin A for 30 min. HeLa and HUVEC cells were pre-incubated with growth medium without antibiotics but supplemented with 20 µM chlorpromazine, 2 µg mL-1 filipin or 100 µM amiloride for 30 min. Then, the GQDs were added to each well (1 mg mL-1 for RAW 264.7 and HeLa cells and 0.5 mg mL-1 for HUVEC cells) and incubated for an extra 4 and 24 h, respectively, at 37 ˚C and 5% (v/v) CO2. Cells exposed to GQDs without inhibitor exposure were used as controls. After the incubation times, the cells were washed 3× in PBS in order to remove non-internalized GQDs. The cells were harvested from the wells for flow cytometry analysis. Raw 264.7 cells were scrapped from the well, while HeLa and HUVEC cells were trypsinized with 60 µL trypsin. The statistical differences between the different conditions were evaluated using the same Tukey´s multiple comparison posthoc test as described in the cell viability assay.


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Scheme 1. a) Synthesis scheme for GQDs synthesis from GO and the actual digital picture with and without 360 nm-wavelength UV excitation. b) Mechanism of the formation of hydroxyl radical (.OH) and the attack on the epoxy groups on GO to synthetize GQDs at hydrothermal (high temperature high pressure; HTHP) condition.


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3. Results and Discussion 3.1. GQDs Synthesis and Optimization In order to use GQDs in real-world applications, a simple, efficient and reproducible green synthesis strategy is highly desirable.27 In the present work, GQDs were synthesized by a singlestep hydrothermal synthesis approach (Scheme 1). GO was chosen as the precursor for the topdown synthesis strategy and synthesized according to the previously reported modified Hummers’ method.31 The prepared GO has an impurity-free regular structure with an average thickness of 0.8-1.2 nm and average lateral dimension of around 1 µm.32 According to previously reported literature, H2O2 can act as a degrading agent for graphene.16 Therefore, H2O2 was chosen as a ‘chemical scissor’ as well as an oxidant for cutting down the GO into smaller sized GQDs. We treated the mixture of GO and H2O2 under hydrothermal reaction conditions at 180 °C. During the reaction, H2O2 was decomposed to form hydroxyl (HO.) and hydroperoxyl (HOO.) free radicals which further converted to oxygen (O2) and H2O. .OH had a standard electrode potential of 2.8 V and it acted as a powerful oxidizing agent.33 The generated .OH radicals attacked the 2D GO sheets from both sides and cut them into smaller sized sheets. Both sp2 and sp3 hybridized carbons were attacked by the .OH radical breaking down both C-C and C=C adjacent to oxygen-containing functional groups. The as-prepared small GO sheets were again attacked by the .OH radicals and converted to smaller sized GQDs.34 The synthesized GQDs displayed a number of defects and a fair amount of oxygen containing functional groups, which were responsible for the electronic transition between the bonding and antibonding level, thereby showing a fluorescent signal under a particular excitation wavelength. In order to achieve uniformly sized GQDs with high optical properties, the reaction conditions needed to be optimized. We focused mainly on three different parameters, namely concentration


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of H2O2, hydrothermal reaction time and temperature. As observed from the obtained reaction products, a reaction time of less than 2 hours (60, 75, 90, 110 minutes) produced a chocolate brown colored solution due to the presence of unreacted GO; while reaction time of more than two hours (130 and 150 minutes) produced a colorless solution due to the further degradation of the GQDs. The reaction products of different concentrations (2, 3, 4, 5 and 6%) of H2O2 also showed a similar effect (Figure S1). As the H2O2 concentration was increased from 2 to 5%, the GQDs solution color turned to clear yellow and started to become faint at a concentration 6% H2O2, which was due to the over-oxidation effect (Figure S1a). In order to optimize the temperature for the GQD synthesis, different temperature conditions were tried (150°C, 160°C, 180°C and 200°C) for two hours. A similar effect was also observed for the temperature change. For the temperature less than 180°C, the presence of unreacted GO were observed, and for the temperature more than 180°C, a colorless solution was produced due to over oxidation effect. Therefore, 5% H2O2 and a two-hour reaction period at 180 °C were chosen as optimized conditions to obtain GQDs with high fluorescent activity. The H2O2 also served as an environmentally friendly oxidant due to its green by-products. The obtained GQDs were freeze dried to obtain a light yellow powder, which further helped to remove the excess amount of H2O2. The reaction yield was measured as 28 ± 1%. As such, this synthesis methodology can be envisaged as a low-cost procedure for the mass production of GQDs.


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Figure 1. a) FTIR spectra and b) XPS spectra of GO and GQDs prepared with different concentrations of H2O2 (2-5%). The high resolution deconvoluted C 1s spectra for c) GO and d) GQDs.

3.2. Structural Characterization of GQDs Structural characterization of the as-synthesized GQDs was performed systematically and the main results are highlighted in Figure 1. As shown in Figure 1a, the Fourier transform infrared (FTIR) spectrum of GO showed the typical characteristic stretching frequencies for different


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functional groups such as hydroxyl O-H (~3400 cm−1), carboxyl C=O (1725 cm−1), alkene C=C (1630 cm−1), epoxy C-O-C (1225 cm−1) and alkoxy C-O (1047 cm−1). After the hydrothermal reaction with increasing concentrations of H2O2, the O-H band at 3400 cm−1 and the C=C band at 1611 cm−1 started to decrease, which further confirmed that GO was degraded into the smallsized GQDs with a lower amount of carbon atoms and oxygen-containing functional groups. Moreover, for GQDs, the peak for epoxy C-O-C groups at 1225 cm−1 disappeared due to the attack of HO. radicals on epoxy groups of GO. Another new peak at 1142 cm−1 provided a clear indication of the presence of C-O stretching vibrations. Due to the presence of these oxygen containing hydrophilic groups, the synthesized GQDs were well dispersed in water. The X-ray photoelectron spectroscopic (XPS) data analysis clearly showed the presence of oxygen and carbon for both GO and GQDs (Figure 1b). The C/O ratio increased with the concentration of H2O2 in the reaction mixture, indicating that more GOs were converted into small-sized GQDs. Although the reaction was done using hydrothermal reaction conditions, the as-synthesized GQDs displayed a fair amount of oxygen-containing functional groups due to the strong oxidation effect of H2O2. Further, deconvolution of the C 1s peak of both GO and GQD showed three different peaks located at 284.7, 286.4 and 289 eV, respectively, corresponding to C-C/C=C in aromatic ring, C-O (epoxy and alkoxy) and O-C=O (carbonyl) functionalities (Figure 1c-d). With the H2O2 assisted hydrothermal reaction processing, the intensities of C1s XPS peak of epoxy and alkoxy were decreased dramatically. This was due to the attack of hydroxyl radicals especially on the epoxy groups on the GO structure. On the other hand, the intensity of the carbonyl group peak was increased gradually, which was a clear indication that more O-C=O groups or carboxylic acid groups were generated during the reaction. The deconvoluted O1s peak at 532 eV from the GQDs confirmed the presence of oxygen-containing


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functional groups (Figure S2). Moreover, the measured zeta-potential of GQDs was negative (10.11±1.2 mV, n = 5), which was due to the presence of negatively charged oxygen containing functional groups.

Figure 2. High-resolution TEM images and the corresponding size analysis of GQDs_5%.

The Raman spectroscopic data is summarized in Figure S3. Two distinct peaks located at around 1363 cm-1 (D-band) and 1592 cm-1 (G-band) were seen for GO. The D band is normally


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associated with structural defects and the G band with the E2g vibration mode of the sp2 carbon region. The intensity ratio of the D to the G band (ID/IG) for GO was 0.92, but for GQDs the value was 0.82, which indicated that the hydrothermal synthesis had introduced more structural defects into the structure of the GQDs. Figure 2 showed a typical transmission electron microscopy (TEM) image of as-synthesized GQDs. According to the size distribution plot, the average size of the GQDs was around 4–6 nm. Furthermore, a well-resolved lattice fringe with a spacing of 0.22 nm was observed from the high-resolution TEM images of GQDs, which corresponded to the d1120 hexagonal lattice plane of graphite.35 This was a clear indication of the high crystallinity of synthesized GQDs. Atomic force microscopic (AFM) image analysis (Figure S5) showed that average thickness of the GQD was around 0.8 – 1.8 nm. The AFM image analysis also agreed with the average size data of GQD from TEM analysis. The X-ray powder diffraction (XRD) analysis was further used to characterize the structure of the synthesized GQDs. As shown in in Figure S4, GQDs had a peak (022) at around 2ϴ = 21.8° and the interlayer spacing is around 0.4 nm, which was broader when compared to graphene (0.34 nm). This was due to the presence of more oxygen containing functional groups in GQDs in comparison to pristine graphene.


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Figure 3. a) UV-vis and FL emission spectra (at 360 nm excitation) of the GQDs. (b) Corresponding FL spectra at different excitation wavelengths ranging from 320 nm to 460 nm (relative FL units; RFU).

3.3. Optical properties The optical properties of the as-prepared GQDs were carefully studied by using UV-vis and FL spectroscopic measurements. As shown in the UV-vis spectra of GQDs (Figure 3a), two peaks at around 230 nm and 300 nm were observed which were due to the π–π* transition of the aromatic C-C bond for the 230 nm peak and the n–π* transition of the oxygen containing functional groups for the shoulder observed at 300 nm. Although, the GO did not show any detectable FL even at basic conditions, the synthesized GQDs exhibited bright blue luminescence in neutral media. Under an excitation wavelength of 360 nm, the aqueous solution of GQDs showed a broad FL peak at 410 nm with a Stokes shift of 50 nm (24.797 eV) (Figure 3a). The appearance broad FL peak was due to the FL band overlap of different oxygen containing functional groups. The FL quantum yield (QY) was calculated to be around 4.6% using quinine sulphate as the standard.36 The as-synthesized GQDs showed an excitation wavelength-dependent FL behaviour (Figure 3b), which was an indication of the involvement of surface defects for the fluorescent


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process, as well as by the ‘Giant red-edge effect’.4 The strongest FL signal was observed at 410 nm for a 360 nm excitation wavelength. When the excitation wavelength was changed from 360 nm to 460 nm, the FL peak shifted to a longer wavelength and its intensity rapidly decreased.

Figure 4. Optical performance stability (FL intensity) of GQDs at different pHs (a), under constant illumination with UV light (360 nm) (b), at different temperatures (c), and upon long term storage (d). (relative FL units; RFU)

The stability of the FL signal was studied under different pH conditions, temperature as well as storage conditions. Four different pH conditions were studied to evaluate the FL of the GQDs.


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As seen in Figure 4a, the FL intensity (relative FL units; RFU) signal increased upon changing from the pH 3 to 7, while a decrease in FL intensity was observed from pH 7 to 10. As confirmed by XPS and FTIR characterization, the GQDs had abundant O-H and oxygen-containing functional groups on the outer edges of each particle. Therefore, most of those functional groups at the edges were protonated at acidic pH and FL quenching occurred.13 The quenching of the FL further indicated the importance of oxygen-containing functional groups for the FL emission of GQDs. As previously proposed by the Wu et al,3 the FL behaviour of GQDs probably originated from the oxygen-containing functional groups at the free zigzag edges with carbene-like triplet ground state. Under strong acidic conditions, the free zigzag sites become reversibly protonated with H+. Therefore, the emissive triplet carbene state could no longer strongly contribute to the FL signal. However, due to their reversible nature, the free zigzag structures were restored again under alkaline conditions and exhibited a marked increase in FL signal. The stability of the GQDs fluorescent signal was further assessed. A photo-bleaching experiment was conducted by constantly irradiating a GQDs sample for 2 hours with UV light at 360 nm, and the FL intensity was measured at regular intervals. As shown in Figure 4b, the FL intensity signal of the GQDs was mostly stable over time with only a 6.3% decrease observed after 2 hours. As depicted in Figure 4c, the FL of the GQDs was slightly influenced by changes in temperature between 4 °C to 25 °C (room temperature). However, a decrease in FL intensity was observed when increasing the temperature to 60 °C. This was probably due to the dissociation of oxygen-containing functional groups at the edges of GQDs at high temperatures. The long-term stability of the as-synthesized GQDs was investigated by storing a single batch of the sample for more than 40 days in a dark environment followed by measuring the FL intensity signal on a


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regular basis. The GQDs solution in water was very stable. As seen in Figure 4d, there was only a 2.1 % decrease in FL intensity even after storing the sample for 42 days. 3.4 GQDs Interaction with Cells QDs have been widely applied in the biomedical field due to their high QY, broad adsorption spectra and excellent stability against photo-bleaching.37 In order to employ the as-prepared GQDs as an effective tool for diagnosis and bio-imaging applications, GQDs should be internalized by the cells while displaying negligible cytotoxicity. We aimed to assess the bioimaging potential of the as-prepared GQDs by studying their interaction with three model cell lines, namely, macrophages (RAW 264.74), which are the first line of defence against harmful organisms, and are found to circulate within almost every type of tissue; endothelial cells (HUVEC), which are the cells lining our blood vessels and, thus, will have significant interactions with intravenous injections of GQDs; and a model cancer cell line (HeLa) which will demonstrate the potential of the as-synthetized GQDs in cancer diagnosis. 3.4.1. RAW 264.7 cells The cytotoxicity of QDs is always an important consideration before their application in cellular or in vivo studies. Thus, we evaluated six different concentrations of GQDs in order to determine the maximum amount of GQDs that could be administered to RAW 264.7 cells without causing a detrimental effect. The measurements were normalized to cells only, that is to say that, cells were incubated without the addition of GQD. Figure 5a shows the normalized cell viability (nCV) results, demonstrating that the maximum concentration of GQDs that could be administered to RAW 264.7 cells was 1 mg mL-1 (Figure 5a, white bars). At this concentration, the nCV readings after 4 h of incubation were higher than 80%. As such, we decided to proceed with this concentration for all subsequent cell association and internalisation assays. Interestingly, in


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contrast to the results observed after 4 h of incubation, when incubating the RAW 264.7 cells for 24 h with a concentration 1 mg mL-1 of GQDs, the nCV readings increased to ~90%, which indicated a potential recovery of the cells when incubated for longer time periods. The FL properties of the GQDs allowed us to monitor and track their cellular uptake. In order to evaluate the cell internalization and association of the GQDs in different cell lines, we assessed the cell mean FL intensity (nCMFI) of RAW 264.7 cells by flow cytometry after 4 and 24 h of incubation (Figure 5bi). The results showed that, after 4 and 24 h of incubation, the nCMFI was ~5 and ~7 times higher than the autofluorescent level of the cells, respectively. These results indicated that the GQDs were successfully associated or internalized by RAW 264.7 cells. The percentage of cells associated with GQDs was also evaluated, and the results demonstrated that ~50% of the cells were associated with the GQDs after 4 h of incubation, a percentage that increased to ~60% after 24 h of incubation (Figure S6a). Next, to confirm that the GQDs were successfully internalized by the RAW 264.7 cells and were not only associated with their membranes; macrophages exposed to GQDs were fixed, the membranes were stained and visualized by FL microscopy by performing a z-stack analysis. As controls, cells without the presence of GQDs were also imaged. Figure 5bii depicts the FL microscopy images which confirmed the GQDs internalization by the cells. As shown by the overlay image (right image) of the stained membranes (TedRex channel, middle image) and the GQDs (DAPI channel, left image) and the corresponding x-z and y-z stack projections, a higher blue fluorescent signal arising from the GQDs was observed after 4 and 24 h of incubation as compared to the control of cells only (Figure S6ii). The FL microscopy images were in agreement with the flow cytometer results, since they also showed a higher fluorescent intensity signal after 24 h of incubation as compared to 4 h of incubation.


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Figure 5. GQDs interaction with RAW 264.7 cells. a) Normalized cell viability (nCV) of RAW 264.7 cells upon exposure to GQDs for 4 and 24 h, respectively. b) i) Normalized cell mean FL intensity (nCMFI) of RAW 264.7 cells upon exposure to 1 mg mL-1 of GQDs for 4 and 24 h, respectively. ii) Fluorescence microscopy images of RAW 264.7 cells incubated with GQDs (DAPI channel) for 4 and 24 h, respectively. The actin filaments of the cells have been stained with phalloidin-TRITC (TedRex channel). c) nCMFI of RAW 264.7 cells upon exposure to GQDs in the presence of chemical inhibitors (i.e., chlorpromazine, filipin, amiloride or latrunculin A). n = 3, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

To elucidate the cell internalization pathway of the GQDs by the RAW 264.7 cell, we investigated the GQDs uptake in the presence of several chemical inhibitors such as chlorpromazine, which inhibits clathrin-mediated endocytosis (CME);38 filipin, which inhibits caveolae-mediated endocytosis (CvME);39 amiloride, which inhibits macropinocytosis;38 and lantruculin A which inhibits phagocytosis.39 Figures 5c and S6c demonstrated a statically significant reduction in both nCMFI and cell uptake efficiency in the presence of chlorpromazine and filipin for 4 and 24h of incubation. Filipin led to a higher decrease of both nCMF and cell uptake efficiency when compared to chlorpromazine, which indicated that GQDs were mainly internalized by CvME followed by CME. Interestingly, although RAW 264.7 cells are specialized macrophages and, thus, are expected to engulf by phagocytosis, Latrunculin A resulted in less pronounced decrease in nCMFI and a non-significant decrease in percentage of cell uptake efficiency. 3.4.2. HUVEC cells For the assessment of interaction of GQDs with HUVEC cells, we first evaluated the nCV of HUVEC cells when exposed to different concentrations of GQDs after 4 and 24 h of incubation time (Figure 6a). The cell viability readings were normalized to cells only. The results


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demonstrated a significant reduction of nCV readings when the cells where incubated with GQDs at a concentration of 0.5 mg mL-1 and 1 mg mL-1 for 4 h and 24 h, respectively. The nCV was ~90% when incubating the cells for 4 h in the presence of GQDs at a concentration of 0.5 mg mL-1, thus demonstrating good biocompatibility. We therefore selected this concentration to conduct the following experiments. To assess the cell association/uptake efficiency of the GQDs with HUVEC cells, we monitored the nCMFI readings after 4 and 24 h of incubation, respectively. As shown in Figure 6bi, the nCMFI was ∼3 and ~4 times higher than the autofluorescent level of cells after 4 and 24 h of incubation, respectively. Figure S7a shows that ~40% of the cells were associated to GQDs or had internalized them after 4 h of incubation, while the percentage increased to ~50% after 24 h of incubation. To confirm that the GQDs had been internalized by HUVEC cells and were not only associated with their membranes, we repeated the same procedure as previously described for RAW 264.7 cells and visualized them by FL microscopy (Figure 6bii). As controls, HUVEC cells incubated in the absence of GQDs were also imaged (Figure S7bii). The blue fluorescent signal arising from the GQDs (as shown in DAPI channel and overlay image) confirmed the internalization of GQDs after 4 and 24 h of incubation, respectively.


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Figure 6. GQDs interaction with HUVEC cells. a) Normalized cell viability (nCV) of HUVEC cells upon exposure to GQDs for 4 and 24 h, respectively. b) i) Normalized cell mean FL intensity (nCMFI) of HUVEC cells upon exposure to 1 mg mL-1 of GQDs for 4 and 24 h, respectively. ii) Fluorescence microscopy images of HUVEC cells incubated with GQDs (DAPI channel) for 4 and 24 h, respectively. The actin filaments of the cells have been stained with phalloidin-TRITC (TedRex channel). c) nCMFI of HUVEC cells upon exposure to GQDs in the presence of chemical inhibitors (i.e., chlorpromazine, filipin or amiloride). n = 3, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

To clarify the cell entry mechanism(s) of the GQDs used by HUVEC cells, the cells were incubated with GQDs in the presence of three inhibitors. Lantruculin A was excluded since phagocytosis is only conducted in specialized cells. Both the nCMFI of the cells (Figure 6c) and the percentage of cells with associated/internalized GQDs (Figure S7c) were analysed. As depicted in Figure 6c, the cell uptake of GQDs was highly inhibited by the presence of filipin, which resulted in a ~50% and ~56% decrease in nCMFI as compared to the cells without inhibitors, after 4 and 24 h of incubation, respectively. The chlorpromazine inhibitor resulted in a reduction of ∼15% of the nCMFI both after 4 and 24 h of incubation, and a similar decrease was observed for the cell uptake efficiency values for both time points (Figure S7c). These results demonstrate that the GQDs were mainly internalized by HUVEC cells by CvME followed by CME at a much lower extent.


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3.4.3. HeLa cells We finally aimed to demonstrate that GQDs could also be internalized by HeLa cells, a cancer model cell line, to highlight the GQDs potential in cancer diagnostics. Therefore, we first evaluated the maximum amount of GQDs that could be administered to HeLa cells without causing a significant decrease in nCV after incubation for 4 and 24 h, respectively. As shown in Figure 7a, there was significant decrease in nCV when HeLa cells were exposed to a concentration of 0.5 mg mL-1 and 1 mg mL-1 of GQDs for 4 and 24 h, respectively. Since the nCV was still ~80% and ~85% after 4 and 24 h of incubation with a concentration 1 mg mL-1, respectively, this concentration was chosen to evaluate the GQDs association/internalisation with HeLa cells. In a similar manner as with the previous cell lines, we evaluated the GQDs uptake/association with the HeLa cells by monitoring the nCMFI for both time points. As shown in Figure 7bi, a ∼3.5 and a ~4.5 times higher nCMFI than the autofluorescent level of the cells was observed after 4 and 24 h of incubation, respectively, indicating that the cells were not saturated with GQDs after 4 h of incubation. These results were in agreement with the cell uptake efficiency values, which show ~50% and ~65% of the cells associated with the GQDs after 4 and 24 h of incubation, respectively (Figure S8a).


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Figure 7. GQDs interaction with HeLa cells. a) Normalized cell viability (nCV) of HeLa cells upon exposure to GQDs for 4 and 24 h, respectively. b) i) Normalized cell mean FL intensity (nCMFI) of HeLa cells upon exposure to 1 mg mL-1 of GQDs for 4 and 24 h, respectively. ii) Fluorescence microscopy images of HeLa cells incubated with GQDs (DAPI channel) for 4 and 24 h, respectively. The actin filaments of the cells have been stained with phalloidin-TRITC (TedRex channel). c) nCMFI of HeLa cells upon exposure to GQDs in the presence of chemical inhibitors (i.e., chlorpromazine, filipin or amiloride). n = 3, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Next, we ensured the internalization of the GQDs exposed to HeLa cells by fixing and staining the cells in a similar manner as described for RAW 264.7 and HUVEC cells followed by their visualization under FL microscopy (Figure 7bii). As controls, cells without GQDs were considered (Figure S8c). As shown by the blue fluorescent signal arising from the GQDs of the overlay (right image) and DAPI channel (middle image) together with the x-z and y-z projection panels, the GQDs had been successfully internalised by the HeLa cells. Next, to elucidate the cell internalisation pathway of the GQDs in HeLa cells, the cells were preexposed to three chemical inhibitors followed by incubation with GQDs for the two time points. The presence of chlorpromazine and filipin resulted in a significant reduction of ~19% and ~55% and of ~15% and ~40% in nCMFI after 4 and 24 h of incubation, respectively. These results suggested that GQDs were preferentially internalized through CvME while CME played a minor role. These results were in agreement with previous reports in which QDs were internalized by cells through endocytic pathways.40,41


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4. Conclusion In conclusion, we demonstrated a simple, one-step, fast and optimized hydrothermal method for the preparation of GQDs from GO with assistance of H2O2. The as-synthesized GQDs showed numerous superior properties such as their uniformly distributed size (4-8 nm), high water dispersibility and excellent optical properties. We have also shown the low cytotoxicity and excellent biocompatibility of the reported GQDs. The application of the as-prepared GQDs as fluorescent nanoprobes has been demonstrated by imaging their internalization by three model cell lines, namely macrophages, endothelial cells and a model cancer cell line, thus demonstrating their potential in bio-imaging and diagnostics. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.XXXXX. Details of graphene oxide synthesis, Comparison with other previously reported synthesis procedures, Fluorescence intensity of graphene quantum dots synthesized in different reaction conditions, Details of XPS, Raman spectral data and XRD data, AFM data, Details of cell uptake efficiency data for three different cell lines.

AUTHOR INFORMATION Corresponding Author *[email protected] (Y.S.)


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ORCID Arnab Halder: 0000-0003-3606-3532 Maria Godoy-Gallardo: 0000-0002-7232-3998 Jon Ashley: 0000-0002-7062-1019 Xiaotong Feng: 0000-0002-2060-7673 Tongchang Zhou: 0000-0002-2305-164X Leticia Hosta-Rigau: 0000-0001-8177-4806 Yi Sun: 0000-0002-0210-4730

ACKNOWLEDGMENT This work was financially supported by the Villum Fonden, Denmark (Grant No. 13153), the Lundbeck Foundation, Denmark (Grant No. R163-2013-15402), and the Danish Council for Independent Research (Grant No. 6111-00298B). REFERENCES (1)

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