Research Article ISSN 1998-0124 CN 11-5974/O4 2021, 14(3): 674–683 https://doi.org/10.1007/s12274-020-3096-y Graphene quantum dots: From efficient preparation to safe renal excretion Caroline Hadad1,2,§, José Miguel González-Domínguez1,3,§, Silvia Armelloni4,¶, Deborah Mattinzoli4,¶, Masami Ikehata4, Akcan Istif1, Adrian Ostric1, Francesco Cellesi5, Carlo Maria Alfieri4,6,7, Piergiorgio Messa4,6,7, Belén Ballesteros8, and Tatiana Da Ros1 () 1 Centre of Excellence for Nanostructured Materials (CENMAT), INSTM, Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy 2 Current address: Laboratoire LG2A – CNRS UMR 7378, Université de Picardie Jules Verne, 33 rue Saint Leu - UFR des Sciences, 80039 Amiens, France 3 Instituto de Carboquímica (CSIC), C/Miguel Luesma Castán 4, E-50018 Zaragoza, Spain 4 Renal Research Laboratory-Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy 5 Dipartimento di Chimica, Materiali ed Ingegneria Chimica “G. Natta”, Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy 6 Unit of Nephrology, Dialysis and Renal Transplant Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via della commenda 4, 20122 Milan, Italy 7 Department of Clinical Sciences and Community Health, University of Milan, 20122 Milan, Italy 8 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and the Barcelona Institute of Science and Technology, Campus UAB, Bellaterra, 08193 Barcelona, Spain § Caroline Hadad and José Miguel González-Domínguez contributed equally to this work. ¶ Silvia Armelloni and Deborah Mattinzoli contributed equally to this work. © The Author(s) 2020 Received: 23 June 2020 / Revised: 3 September 2020 / Accepted: 6 September 2020 ABSTRACT Carbon nanomaterials offer excellent prospects as therapeutic agents, and among them, graphene quantum dots (GQDs) have gained considerable interest thanks to their aqueous solubility and intrinsic fluorescence, which enable their possible use in theranostic approaches, if their biocompatibility and favorable pharmacokinetic are confirmed. We prepared ultra-small GQDs using an alternative, reproducible, top-down synthesis starting from graphene oxide with a nearly 100% conversion. The materials were tested to assess their safety, demonstrating good biocompatibility and ability in passing the ultrafiltration barrier using an in vitro model. This leads to renal excretion without affecting the kidneys. Moreover, we studied the GQDs in vivo biodistribution confirming their efficient renal clearance, and we demonstrated that the internalization mechanism into podocytes is caveolae-mediated. Therefore, considering the reported characteristics, it appears possible to vehiculate compounds to kidneys by means of GQDs, overcoming problems related to lysosomal degradation. KEYWORDS graphene quantum dots, podocytes, biodistribution, uptake pathway, renal clearance, nanocarrier 1 Introduction then-called “nano graphene oxide” showed unprecedented biocompatibility and practical value, being the first application Since 2004, the landmark study by Geim and Novoselov drew example of what is known today as graphene quantum dots the attention into graphene, that presents undisputed potential (GQDs). in many areas, particularly in optoelectronics, due to its GQDs consist of tiny crystalline zero-dimensional pieces of unique electronic structure and overall physical properties [1]. graphene, generally oxidized in a certain degree, receiving their Its oxidized derivative, graphene oxide (GO) is also largely designation from the analogy with traditional semiconductor popular for its high hydrophilicity, easy handling and affordable quantum dots (e.g. CdSe). Their small size is enough to cause production. Also in this case there is an interesting niche of quantum phenomena by exciton confinement and edge effects, applications, as electrochemistry [2] and biotechnology [3] resulting in photoluminescent (PL) properties. Graphene and from the thorough study of GO and related derivatives, exhibits a zero electronic bandgap, but a fragment of graphene novel discoveries emerged. In 2008 Sun et al. [4] realized (technically of any size if small enough) will display a non-zero on the potential of nanometric-sized flakes of GO for bandgap leading to luminescence upon excitation [2]. This biomedicine, as these possessed luminescent properties coupled bandgap can be tailored through the GQDs geometry, size and to multifunctionalization possibilities for both cellular imaging chemical composition of their edges [5], giving rise to a wide and anticancer drug delivery in specific cell lines. This back- range of possible emissions. Unlike the classical semiconductor Address correspondence to daros@units.it Nano Res. 2021, 14(3): 674–683 675 quantum dots, GQDs are intrinsically water-soluble and rather (rGOQDs) were tested and a dose-dependent variation of the easy to handle at a chemical level, since they can be treated animal development was reported [18]. Analyses on zebrafish almost as molecules, and not as unstable/metastable colloids, larvae at different concentrations (12.5–200 μg/mL) resulted opening many venues for their study. in embryo malformations with the higher concentrations The rapid rise of these nanostructures in recent years all treatment, while the lower resulted to be safe [19]. To evaluate over the world has generated different nanostructures termed the biodistribution also radioactive tracking was used. Carbon as GQDs or GO quantum dots (GOQDs) with slight variations materials obtained by the pyrolysis of citric acid were in chemical composition and shape, mostly depending on functionalized using Na131I. Also in this case, despite the different their synthesis route and nature of the carbon precursor. This source and decoration of the materials, the fate was the same, diversity would need a systematization of nomenclature according with higher concentrations in liver and kidney, suggesting renal to the presence of the graphene lattice (to properly distinguish excretion. Moreover, in tumor bearing mice, the concentration GQDs from carbon dots), the lateral size, the thickness and in the affected tissue was quite good [20]. degree of oxidation in a similar way as done for graphene [6], in Despite all these encouraging results, the toxicity of GQDs order to delimit the structural and compositional conditions is an important subject, which must be further addressed, since in which the quantum confinement stands. However, until there is still a lack of knowledge for many unexplored cell lines that happens, scientists will tend to use the terms GQDs and and in vivo models and, moreover, the studied materials often GOQDs indistinctly. With a lateral size ranging from few differ for chemical and physical characteristics. In particular, nanometers to 100 nm (typically up to 20 nm), and in the form it would be of a great interest to confirm their biodistribution of one or few layers, all less than 10 nm, these carbon nanoforms in vivo in more models and to unravel the exact excretion can be obtained via top-down or bottom-up approaches [6–8]. mechanisms in view of a future application of GDQs in The top-down methodology seems to be the most followed for bioimaging or nanomedicine. the production of GQDs, as it usually grants larger-scale outputs The present work describes the reproducible top-down besides being highly versatile and readily accessible [9, 10]. synthesis of GQDs starting from GO with a nearly 100% The choice of the carbon precursor determines many features of conversion of the starting material. Cytotoxicity of the obtained the eventual nanostructures, such as its shape [9], heteroatom GQDs was then assessed in vitro on human renal cells, and in doping [11], and photophysical properties [12], hence a huge mice. Indeed, vascular endothelial cells, glomerular basement diversity of feedstocks have been reported in literature for this membrane (GBM) and podocytes represent the main com- purpose, including biomass wastes, mineral carbons, carbon ponents of glomerular filtration barrier, whose alteration nanostructures, synthetic polymers and biological entities [7]. compromise renal function and results in proteinuria, a sign As regards to their PL properties, GQDs display a typically of glomerular disease [21]. For this reason, these cells constitute broad emission dependent on the excitation wavelength, and an appealing target for nanoparticles (NPs) drug delivery. A these can be easily tuned, apart from the GQDs own features, model of glomerular filtration barrier, composed of endothelial throughout pH, dispersion medium or even upon post- cells, collagen IV, a protein of GBM, and podocytes, were used synthesis surface modification [12]. to examine GQDs passage [22]. Moreover, the evaluation of GQDs possess an unparalleled biocompatibility, chiefly cytoskeletal proteins distribution was assessed in podocytes stemming from their high water-solubility, reason why these after NPs addition; F-actin represents an important marker to nanostructures are intensively applied in biological or biomedical assess the maintenance of the correct cellular morphology and studies, showing exceedingly higher biocompatibilities than the functional integrity [23, 24] and was used to verify GQDs semiconductor quantum dots, even in the absence of chemical biocompatibility. derivatization [13]. Some works dealing with bioimaging, Podocytes are highly differentiated cells characterized by biosensing, and disease treatments are reported in literature, a large cytoplasm with primary and secondary processes, suggesting that GQDs have excellent biocompatibility, with protruding and interconnecting [25]. As known, size and charge relatively low in vitro and in vivo toxicity [14]. Overall, GQDs of biomolecules determine NPs repulsion or absorption by the seem much less toxic than GO as reported in many studies. In barrier [26]. Only NPs below 80 nm pass the endothelial this context, Chong et al. described the synthesis of GQDs by fenestration and lower than 32 nm the ultrafiltration barrier a graphite powder modified cutting method and found that, of podocytes, consisting of a bridge between the secondary unlike GO, which tends to form aggregates, no obvious in vitro processes. The impairment of these components may occur and in vivo toxicity was found, even under a multi-dosing during primary and acquired nephropathies and the delivery situation [15]. of the drugs directly on damage cells, may constitute the goal Not many papers reported about the biodistribution of of GQDs use in human therapy. GQDs and one of the first was by Chong and co-workers [15]. Other experiments on GQDs obtained by an electrochemical 2 Experimental method and presenting dimensions in the range of 160–280 nm, were performed, in which the GQDs were directly radiolabeled 2.1 Material and methods with 99mTc and used for in vivo studies on mice and for in vitro mutagenicity tests. While from the in vitro experiments some 2.1.1 Reagents, materials and instruments mutagenic activity emerged with changes from A:T to G:C, Chemical part the in vivo experiments resulted in good biocompatibility and an equal biodistribution in healthy mice organs. Instead, the Mineral acids, used at higher commercially available concentration, case of endometriosis model mice, a strong increase in the were from Sigma-Aldrich (Milan, Italy) or Alfa Aesar (Thermo uptake by kidneys and of the renal clearance was reported [16]. Fisher, Milan, Italy). Dialysis sacks were from Sigma Aldrich To exert the in vivo safety, studies were also performed on D6066. High purity graphite powder was from Carbon Bay zebrafish embryos, which properly grew despite the GQDs (grade SP1, batch #04100, lot #011705). Milli-Q water (resistivity accumulation in the digestive tract, tracked using the GQDs of 18 MΩ·cm), obtained from a Millipore system, was used in photoluminescence [17]. Analogously, reduced GOQDs all experiments and cleaning steps. thermogravimetric analysis www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 676 Nano Res. 2021, 14(3): 674–683 (TGA) was recorded on a TGA Q500 (TA instruments), under spectroscopy measurements were performed using a Xenius N2 flow, by equilibrating at 100 °C, and following with a ramp SAFAS spectrophotometer. at 10 °C/min up to 800 °C (approximately 1 mg of each com- pound). High-resolution transmission electron microscopy 2.2 Procedures (HR-TEM) and HAADF-STEM images and energy-dispersive 2.2.1 Synthesis of graphite oxide (GO) X-ray spectroscopy (EDX) spectra were acquired using a FEI Tecnai G2 F20 microscope operated at 200 kV coupled to an GO was obtained according to the modified Hummer’s method. EDAX super ultra-thin window (SUTW) X-ray detection system. 3.11 g of graphite powder and 1.54 g of sodium nitrate were Samples were prepared by drop casting from the GQDs aqueous added into 70 mL concentrated H2SO4 at 0 °C and stirred for solution onto a TEM grid (300 mesh, ultrathin carbon on holey 30 minutes. After this time, KMnO4 (9.06 g) was gradually carbon coated copper grid). Infrared spectra were obtained added to the mixture under continuous stirring in ice-bath by using a Fourier-transform infrared (FT-IR) spectrometer for 2–3 minutes. Then, the mixture was heated at 35 °C for (Perkin-Elmer 2000) through pellets with spectroscopic 30 minutes and 138 mL of water were added. The temperature grade KBr. The ultraviolet–visible–near-infrared (UV–Vis–NIR) of the mixture was increased until 100 °C and maintained for spectroscopy measurements were carried out with a Varian Cary 15 minutes. The obtained brown solution was diluted into 5000 spectrophotometer by using 1 cm path length polystyrene 420 mL of water under stirring and the reaction was quenched cuvettes. Fluorescence emission spectra have been recorded after addition of 3 mL of H2O2. Then, the stirring was on a Varian Cary Eclipse Spectrophotometer using 10 mm switched-off, the graphite oxide (brown suspension) settled to path length Hellma Analytics 117.100F-QS quartz cuvettes. the bottom overnight and the orange supernatant was decanted For atomic force microscopy (AFM) visualization, the samples off. 200 mL of water were added to the graphite oxide and the were prepared by drop-casting the GQDs aqueous solution onto mixture was stirred for 5 minutes. After 1 h of settlement, the SiO2/Si wafer substrates and dried under vacuum. Measurements graphite oxide deposited in the bottom and the supernatant were performed with a Nanoscope V (Digital Instruments was removed. This operation was repeated 3 times. 100 mL Metrology Group, model MMAFMLN) in tapping mode in of water were added to the brown deposit and stirred for air at room temperature, using an n-type silicon μmash® SPM 10 minutes. The mixture was then transferred into dialysis probe (HQ:NSC15/AL BS) with tip height 12–18 μm, cone sacks against Milli-Q water until pH 4–5 and then lyophilized angle <40° (Resonant frequency 325 kHz, force constant to obtain 2.85 g of GO. of ~ 40 N/m). The collected images were then analyzed 2.2.2 GQDs preparation with WsXm 4.0 software (Nanotec Electronica S. L.), and Gwyddion 2.39. Dispersions of GO in sulfonitric mixture In a typical experiment, 6 sealed glass vials of 20 mL containing were sonicated in a temperature-controlled bath sonicator 10 mg of GO and 2.5 mL of acidic mixture (concentrated (Elmasonic S, 37 kHz). H2SO4/HNO3 mixture, 3:1) were sonicated for 24 h at 80 °C using a sonic bath (if necessary, vial caps were replaced every Biological part 8 h of sonication). After this time elapsed, vials were removed Hemangioendothelioma Endothelial Cells (EOMA) (ATCC® from the sonic bath, allowed to cool down to room temperature CRL-2586™) were from LGC Standards S.r.l. (Sesto San and the dark brown liquids contained in each vial were carefully Giovanni, Milan, Italy). Podocytes SV1 cell line, from transgenic diluted with Milli-Q water until the top of the vial (8-fold H-2Kb-tsA58 mice, were from Cell Line Service (Eppelheim dilution) and hand-shaken for a few seconds. After settling Germany). 96-well plates used to grow cells were from Corning down at room temperature overnight, hand shaking was again (Milan, Italy). All reagents used for culture maintenance were performed and the orange solutions were gathered together from Sigma-Aldrich as well as Trypan blue staining phalloidin and transferred into dialysis sacks. The dialysis against Milli-Q 488 probe and 4',6-diamidino-2-phenylindole (DAPI). The water took place with frequent water replacements, until the LDH-Cytotoxicity Colorimetric Assay Kit was purchased by pH of the outer waters became neutral. It is important to Bio Invision Incorporated Vinci-Biochem (Firenze, Italy), the mention that a careful and thorough tracing of the dialysis tetramethylrhodamine-albumin bovine serum by Invitrogen, process was undertaken, and no fluorescence was ever noticed while paraffin was from Histo-line laboratories (Milan, Italy), in the dialysis waters, neither carbonaceous solids upon filtrating optimal cutting temperature compound by Sakura Finetek them, meaning that GQDs fully remained inside the dialysis Europe B.V. (Netherlands), and Carazzi’s Hematoxylin-Eosin sacks. Once neutralized, the resulting yellow solution of GQDs from Bio-Optica (Milan, Italy). Polyethylene terephthalate (0.22 ± 0.04 mg/mL) was taken out from the sacks, protected (PET) membrane 1 μm porous (Millicell hanging cell culture from the light and stored at 3–4 °C until further characterization inserts) was from Millipore (Milan, Italy). Mice C57BL/6N or use. By freeze-drying this solution, a spongy and fluffy light were from Charles River Laboratories (Calco, Lecco, Italy). solid was obtained and used for TGA and IR analyses. Coverslips were supplied by Thermanox, Nunc, VWR Int. 2.2.3 Mouse podocytes and hemangioendothelioma endothelial (Milan, Italy). cells line culture Absorbance was obtained by Xenius SAFAS spectrophotometer (Safas, Monaco). Immunofluorescence images were acquired by EOMA were maintained in incubator at 37 °C at 5% CO2, in Zeiss AxioObserver microscope equipped with high-resolution Minimum Essential Medium Eagle (EMEM) supplemented digital videocamera (AxioCam, Zeiss) and Apotome system with 10% fetal bovine serum and 1% penicillin/streptomycin, for structured illumination and recorded by AxioVision software according to manufacturer’s protocol. Podocytes were previously 4.8. Histological slices were observed by Zeiss Axioscope 40 expanded at 33 °C in DMEM:F12 medium supplemented with microscope equipped with AxioCam MRc5 digital camera. 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM Histological images were then acquired by Zeiss AxioObserver L-glutamine and 20 U/mL recombinant mouse γ interferon. microscope equipped with high resolution digital videocamera Subsequently the podocytes were grown in culture flasks (AxioCam, Zeiss) and Apotome system for structured illumination precoated with collagen type IV in a complete medium, without and recorded by AxioVision software 4.8. Fluorescence gamma interferon and thermoshifted to 37 °C for 7 days | www.editorialmanager.com/nare/default.asp Nano Res. 2021, 14(3): 674–683 677 of differentiation. All experiments were then conducted on 488 probe and the nuclei were stained by DAPI. differentiated podocytes. 2.2.7 Animal model and ethics statement 2.2.4 Cytotoxicity assessment by LDH assays All procedures involving animals were performed in accordance For the cytotoxicity assessments, podocytes and EOMA cells with the European legislation (European Communities Council were seeded at 104 cells/cm2 in triplicate into 96-well plates in Directive of 24 November 1986, 86/609/ EEC) and complied a basal medium till 60% of confluence. Lactate dehydrogenase with the European Communities Parliament and Council assays (LDH) were then performed on both SV1 podocytes Directive of 22 September 2010 (2010/63/EU) and with the and EOMA cells according to manufacturer’s instructions. A Italian (D.L.vo n. 26/2014) and the Protocol N. 07/13 approved concentration ranges (10 to 100 μg/mL) of GO and GQDs, for by the Milan University Institutional Care and Ethical Treatment 24 hours at 37 °C have been used. As positive control, 10 μL of Committee. Eighteen mice C57BL/6N of 3 months of age were lysis solution was added for the same time. At the end of housed in plastic cages (5 mice/cage), according to the current incubation, the plate was gently shacked and centrifuged at ethical and legislative rules Directive 2010/63/EU for animal 600g for 10 minutes. Supernatant (10 μL) was then transferred experiments. They were maintained under controlled conditions into new 96 well-plate and the reaction mix was added to each of humidity, temperature and 12 hour light/dark cycles; all well and incubated at room temperature for 1 hour. After animals allowed free access to sterile pelleted food and water adding stop solution, the absorbance was measured at 450 nm ad libitum. and the value of background signal at 680 nm was subtracted. 2.2.8 Evaluation of in vivo GQDs biodistribution 2.2.5 Assessment of GQDs permeability through glomerular Animals were then randomly divided in groups of three and filtration barrier intravenous injections (IV) of 200 μL of GQDs solution at the The use of a three-dimensional model of glomerular filtration final concentration respectively of 10, 25 and 50 μg/mL were barrier allowed assessing the GQDs passage through the performed. For each group three mice were injected with artificial barrier. As previously described by some of us [22], 200 μL of vehicle as control. After 24 h mice have been after coating on both sides PET membranes insert with Type sacrificed after anesthesia induced by intraperitoneal injection IV collagen, EOMA cells were seeded on the lower side of the of 370 mg/kg of chloral hydrate and brain, liver, kidney, lung, membrane and cultured for 1 week in the presence of 5 ng/mL spleen, stomach, colon, and bladder were excised and urine of VEGF. Once cells attached, podocytes were seeded on the collected for further investigation. opposite side of the membrane and incubated in their own 2.2.9 Histological analyses complete medium for differentiation. Each insert was placed Brain, liver, kidney, spleen, lung, stomach, colon, and bladder in a 24-well plate with the EOMA side facing the bottom of were embedded in paraffin and optimal cutting temperature the well. GQDs 30 μg/mL were added to EOMA side in each compound. Paraffin sections (5 μm thick) were de-waxed in well. At different time point (30 minutes, 1, 2, 4 and 24 hours) xylene and rehydrated in gradient alcohol for 5 minutes. Carazzi’s medium on the side of podocytes was collected and optical Hematoxylin-Eosin staining were performed according to density was measured by spectrophotometer at 550 nm of standard protocol. Sections were then observed under excitation and 570 nm of emission respect to a GQDs microscope. 5 sections were prepared for subsequent analysis. concentration curve. The time dependent curve obtained was Frozen tissue (4 μm thick) were fixed in PFA 4% for 10 seconds then compared to the tetramethylrhodamine-albumin bovine and stained with DAPI to visualize the nuclei, images were serum (TRITC-BSA) curve detected at the same wavelength. acquired for tissue GQDs distribution determination. The BSA permeability, used as control, was obtained adding 40 mg/mL of TRITC-BSA to the cells instead of GQDs. The 2.2.10 GQDs quantification in the urine kinetic and the amount of GQDs and BSA that permeated Upon injection, each mouse was housed in a metabolic cage to through the glomerular-like artificial filtration barrier were collect 24 hour urine. Controls and treated mice urines were compared. diluted with deionized water to a final volume of 1 mL and 2.2.6 Evaluation of GQDs cellular uptake 100 μL were used to measure the emission by fluorescence spectroscopy. Podocytes (20,000 cells/cm2) were grown on coverslips at 50% of confluences, the cells were then treated with 30 μg/mL of GQDs and observed after 30, 60 and 90 minutes. In the aim to 3 Results deepen the uptake pathway, podocytes were pre-incubated for 30 minutes with different endocytosis inhibitors at concen- 3.1 Synthesis and characterization of GQDs trations previously tested from some of us [27]: filipin III 1 μg/mL, Graphite oxide was first obtained from graphite powder using genistein 200 μM, wortmannin 100 nM, chlorpromazine the so-called Hummers method [28]. This method is by far hydrochloride 10 μg/mL, sodium azide 10 mM. Three different the one of the most widely used for graphite oxidation, which experimental conditions were used: control with basal medium, involves the use of concentrated acids and the presence of a inhibitors in basal medium and inhibitors followed by of GQDs. strong oxidizing agent. After purification steps mainly based Trypan blue staining vitality test was performed after the on dialysis against Milli-Q water, the resulting material was incubation with the selected inhibitors to reconfirm the cell used as a source for the synthesis of GQDs. Therefore, GQDs death below 50%, after 150 minute of exposure. The amount of in aqueous solution were obtained after sonication of GO in quantum dots uptake was assessed through the quantification a mixture of concentrated acids (i.e. sulfonitric mixture) at of the GQDs fluorescence on 5 fields. 80 °C for 24 h (Fig. 1). After dilution, the presence of water To evaluate a possible cytoskeleton damage after treatment, helps the stabilization of the GQDs in acid environment, being the cells were fixed with 4% PFA for 10 minutes at each time the excess of acids subsequently eliminated by dialysis until point. F-actin cytoskeletal morphology of podocytes was then reaching neutral pH. The concentration of GQDs in water investigated by a direct immunofluorescence with phalloidin was determined after lyophilization of different aliquots. By www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 678 Nano Res. 2021, 14(3): 674–683 Figure 1 Schematic representation of the GQDs synthesis. Procedure process to prepare GQDs. freeze-drying of the obtained solution, the concentration of GQDs was determined as 0.22 ± 0.04 mg/mL and their production was virtually of 100%, since no traces of GO were Figure 2 Characterization of GO and GQDs. (a) TGA analyses of GO found anywhere along the process, as it was checked by and GQDs under nitrogen atmosphere. (b) FT-IR spectra of GO and UV–Vis and atomic force microscopy (AFM) analyses of the GQDs, KBr pellets. (c) Raman spectra of GO and GQDs, λ = 532 nm. GO: elutes, and the described methodology did not involve any black line; GQDs: red line. size-separation step. Both GO and GQDs were characterized using TGA, Raman and IR spectroscopies, TEM, AFM and, in the case of the GQDs, EDX. TGA performed under nitrogen atmosphere (Fig. 2(a)) showed that GO and GQDs are thermally unstable and started to lose mass upon heating above 100 °C, with a significant drop in mass at 212 °C (around 30% of the total mass) for GO and at 164 and 202 °C for GQDs (around 50% of the total mass) corresponding to the thermal desorption of the labile oxygen-containing groups (e.g. carboxylic, hydroxyl or epoxide groups). The oxidation of graphite to GO was also confirmed by FT-IR spectroscopy (Fig. 2(b)). The spectra show the characteristic bands at 3,400, 1,731 and 1,647 cm−1 assigned to the O–H group stretching vibrations and to C=O stretching of carboxylic and/or carbonyl moiety functional groups respectively but also two peaks at about 1,238 and 1,072 cm−1 corresponding to the C–O stretching vibrations. The Raman spectra (Fig. 2(c)) showed the main features of graphitic carbon-based materials with the G and D peaks at around 1,609 and 1,349 cm−1, corresponding to sp2 vibrations and to the disorder-induced by the defects on the sp2 hybridized hexagonal sheet of the carbon, respectively. The morphology of GO and GQDs was investigated through Figure 3 HR-TEM images of GQDs. ((a) and (b)) HR-TEM images of microscopic observation. In the case of GO, large single- or GQDs, size distribution histogram derived from HR-TEM images (inset few-layer sheets were observed in TEM, reaching several in (a)) and image of the crystalline graphene structure, with 0.21 nm micrometers in lateral size (see Fig. S1 in the Electronic interplanar spacing corresponding to the (100) planes (inset in b)). (c) Supplementary Material (ESM)). These large sheets of graphene High-angle annular dark-field (HAADF)-STEM image of GQDs. (d) EDX oxide were cut during the synthesis of GQDs ending up in an spectrum of GQDs. Scale bars: ((a) and (c)) = 20 nm, (b) = 5 nm. The average diameter of 2.9 ± 0.8 nm determined by HR-TEM distribution histogram is based on analysis of 102 GQDs. (Fig. 3). Preliminary DLS measurements of the lyophilized material dispersed in water at a concentration of 100 μg/mL arise from the H2SO4 used for their preparation. The copper resulted in a hydrodynamic radius of 26.2  1 nm (Fig. S4 in signal comes from the TEM grid. the ESM). From HR-TEM, interplanar spacings of 0.21 nm, The AFM characterization was used to ascertain the number corresponding to the (100) plane in GQDs, were observed in of layers of which GQDs were made of, as appreciable from some of the particles (Fig. 3(b), inset). EDX analysis on the Fig. 4 with different magnifications of a deposited sample of GQDs showed the characteristic peaks for carbon and oxygen, GQDs, revealing that their height ranged between 1 and 2 nm as expected, and also a small amount of sulfur, which might (mostly below 1.5 nm). | www.editorialmanager.com/nare/default.asp Nano Res. 2021, 14(3): 674–683 679 for both permeability assay and cellular uptake experiments. 3.3 Analysis of GQDs in vitro renal filtration To better understand whether GQDs cause a difference in renal clearance or damage the filtration barrier, an in vitro test of glomerular filtration was performed. As described by some of us [22], endothelial cells and podocytes, grown on collagen IV-coated membrane, reflect the physiological resistance to albumin permeability [29]. The BSA permeability was then assessed and the curve reached the plateau after 2 h reconfirming our previous result. The same assay was subsequently used to establish the time-dependent permeability of GQDs and the trend was compared to BSA curve. It was possible to assess that GQDs, at the concentration of 3 mg/dL, were able to permeate the filtration barrier but showed a lower passage through the barrier compared to BSA (Fig. S6 in the ESM). 3.4 Evaluation of GQDs cellular uptake The different physical and chemical properties of nano- materials lead to various kinds of interaction with biomolecules and cells [30]. It is therefore important to understand the Figure 4 AFM images of GQDs. Different AFM images of GQDs with preferential GQDs cellular uptake pathway on podocytes [27]. their respective height profiles along the drawn line in each image. The inhibitors concentration (filipin III 1 μg/mL, genistein 200 μM, wortmannin 100 nm, chlorpromazine hydrochloride The typical fluorescence emission from the GQDs (Fig. S2 10 μg/ml, sodium azide 10 mM), already tested by some of us in the ESM, in blue) presents a broad emission band in the visible [27], was reassessed by trypan blue vitality test (Fig. S7(a) in range, with a maximum at 570 nm, implying yellow/green the ESM). The maintenance of the podocyte’s cytoskeletal emission, upon excitation with a 330 nm wavelength, which integrity by F-actin immunostaining was monitored after 30, was determined as the optimum. up to 150 minutes of treatment and no loss of integrity was found after the inhibitor addition compared to the control 3.2 GO and GQDs in vitro cytotoxicity on heman- (Figs. S7(b)–S7(f) in the ESM). gioendothelioma endothelial cells and podocytes The presence and the distribution of GQDs in podocytes To compare the cytotoxicity of GO and GQDs, LDH assay were detected and quantified after 60 minutes. It could be was performed on both podocytes and EOMA with several observed that GQDs were taken up by podocytes and retained concentrations and different time-points. LD50 was calculated in both cytoplasm and nucleus. This internalization was partially and GQDs resulted less toxic than GO at every time point (Fig. impaired after the inhibitor’s addition. In particular, sodium S5 in the ESM). azide (Fig. 5(b)) partially inhibited cellular uptake as also For EOMA, GO LD50 varied from 32 to 47 μg/mL and for wortmannin (Fig. 5(c)), whereas chlorpromazine hydrochloride GQDs from 59 to 63 μg/mL, between 24 and 72 hours of did not inhibit endocytosis of GQDs (Fig. 5(d)). On the contrary incubation. LD50 value of podocytes varied from 32 to 35 μg/mL absence of GQDs was subsequent to filipin III administration after GO incubation and from 40 to 52 μg/mL after GQDs (Fig. 5(e)), as also the marked cytoplasmic reduction following addition at the same time points. These preliminary data genistein (Fig. 5(f)). The data were confirmed by fluorescence indicated that 30 μg/mL was the more suitable concentration quantification (Fig. 6). Figure 5 Diagram of uptake internalization. Diagram of GQDs (red) uptake internalization in the podocytes cytoplasm and the nucleus (DAPI in blue) after inhibitor addition compared to (a) the ctrl, t = 60 min; (b) sodium azide; (c) wortmannin; (d) chlorpromazine hydrochloride; (e) genistein; (f) filipin III. Scale bar 50 μm. www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 680 Nano Res. 2021, 14(3): 674–683 Figure 6 GQDs fluorescence in podocytes. GQDs fluorescence intensity measured in podocytes after the inhibitor addition after 60 minutes. 3.5 Evaluation of GQDs biodistribution in vivo Single-dosing experiments were conducted in mice to assess the in vivo toxicity and biodistribution. Prior fluorescence examination of GQDs distribution, hematoxylin/eosin staining of the organs collected after treatments was performed. First, as expected, no mouse death was observed [31–33], then no clear difference compared to the control animals (Figs. 7(a)–7(e)) Figure 8 GQDs tissue distribution. GQDs (yellow-green) distribution was found in their organ structure (Figs. 7(f)–7(j)). detected by immunofluorescence in control and treated mouse tissues The distribution of GQDs was evaluated in mice kidney, after 24 hours at 25–50 μg/mL. Nuclei DAPI staining (blue). Scale bars 50 μm. spleen, liver, brain, lung, colon and bladder (Fig. 8). When treated for 24 h with GQDs 25 and 50 μg/mL, no distribution was observed in the kidney, both in the glomerulus and in the tubule (Figs. 8(b) and 8(c)). In liver no GQDs were detected at 25 μg/mL, while some NPs were seen after 50 μg/mL treatment (Figs. 8(e) and 8(f)). The compound also accumulated in brain, as reported elsewhere [34] (Figs. 8(h) and 8(i)) and in spleen red pulp at the highest concentration of GQDs (Figs. 8(k) and 8(l)). In lung, bladder, stomach and colon, at both concentrations no GQDs were detected (data not shown) and their presence in urine was evaluated to establish the mice renal clearance (Fig. 9). At the concentration of 50 μg/mL, the amount of GQDs eliminated was less than at the lower concentrations of 25 and 10 μg/mL (Fig. 9). Figure 9 GQDs urine excretion. GQDs quantification in urine of treated (50 and 25 μg/mL) and control mice by fluorescence. 4 Discussion The reported method to prepare GQDs allowed to obtain tiny nanoparticles with mean diameter of about 3 nm. It is important to note that the yield of GQDs production is often quite low, typically below 10%, and rarely reaching 20% or more, so there is a major challenge not only in improving the production scale but also in maximizing the synthesis yield and in this case the conversion of GO to GQDs has been virtually 100%. HR-TEM images of an individual particle demonstrate the crystalline structure of the GQDs, where the 0.21 nm interplanar spacing corresponding to the (100) planes can be observed. The so prepared material was first studied to determine its cytotoxic profile in comparison to GO on endothelial cells and podocytes, main actors of ultrafiltration barrier, since it represents the more useful prerequisite for renal biosafety. In fact, despite many papers reported about the good biocompatibility of GQDs, the differences of the materials due to the preparation methodology render necessary to explore at Figure 7 Tissue staining. Hematoxylin/eosin staining of mice tissues first their cytotoxicity. LDH assay and DAPI nuclear staining after 24 h GQDs injection. Scale bar 50 μm. demonstrated that, in the adopted experimental conditions, | www.editorialmanager.com/nare/default.asp Nano Res. 2021, 14(3): 674–683 681 over 75% of EOMA, about 65% of podocytes survive, with experiments in mice were performed and the histological nuclei without fragmentation. absence of morphological changes confirms the good tolerability These data consequently show a safe profile of GQDs in of GQDs. renal cells, up to the concentration of about 59 μg/mL for As mentioned, not so many in vivo studied have been EOMA and up to 40 μg/mL for podocytes. These results are reported so far. Among the most significant ones, Chong et al. perfectly comparable with the studies of GQDs interaction demonstrated that small size GQDs, rich in oxygen, exert very with macrophages and neurons [34, 35] but show a greater low cytotoxicity and that in mice no accumulation was detected toxicity than that manifested on MGC-803 gastric cancer cells in the principal organs, while a fast excretion through kidney and on MCF7 breast cancer cells [36]. In vitro studies on was reported [15]. Other authors took advantage of the intrinsic Human Adipose-Derived Stem cells (hADSCs) by Kim et al. fluorescence of GQDs to evaluate their biodistribution. In the confirmed the safety of this material with the cell viability case of carboxylated carbon dots, obtained by exfoliation of higher than 80% even when the treatment was performed carbon nanofibers, the material fate was followed by fluorescence using a 2.0 mg/mL concentration of GQDs. Considering these after injection in the tail vein of KB tumor bearing mice. The results, the researchers used them to tag stem cells and to follow materials tend to accumulate in liver, kidney and tumor tissues their fate in vivo, proving the possibility to use this material in without inducing morphological changes as demonstrated by stem cell therapy imaging [37]. Lower concentrations were ex vivo analyses [31]. Also post synthesis functionalized used for studies on rat Schwann cells (RSC96) (10–100 μg/mL) polydopamine-coated GQDs were studied in mice and, after and also in this case the cell viability was above 90% [38] and tail vein injections, the material was distributed to heart, liver, again the GQDs were also used for in vivo imaging, without lung, spleen and kidney and excreted by the latter [41]. tissue autofluorescence interference [36, 39]. Our results are in In our case, the GQDs distribution and storage in various line also with these reports, taking into consideration not only organs was investigated to understand the post-intravenous the difference in the studied materials, but also the differences administration behavior and the clearance pathways. GQDs in the used cell models. Moreover, the herein reported results presence was detected in liver and in spleen, due to their specific demonstrated that the safety of GQDs overcomes that of GO, role in blood multiple metabolites trafficking. In addition, as reported elsewhere [39]. The renal cells that assemble in the their deposition was detected in brain, demonstrating the glomerulus have the characteristic of forming a special barrier capacity to cross blood-brain barrier, as already reported by composed of endothelial cells, GBM and podocytes. The in Kim et al. [34]. Moreover, the absence of GQDs in the vitro results show that the GQDs kinetics curve, in the passage glomeruli and in the renal tubules suggests the lack of through this ultrafiltration barrier, is comparable to that of storage and the rapid leakage from the kidney. The renal BSA and that the greatest amount of GQDs goes through it excretion, reported by Chong and coworkers [15], was studied in few hours without causing damage. After this time, the also for flakes of GO, presenting no more than 3 sheets quantity of GQDs that cross the membrane remains constant. on height with average lateral dimension lower than 1 μm, The GQDs capacity to overcome in vivo the ultrafiltration demonstrating that it takes place without changing or damaging barrier after 24 h was confirmed by fluorescence images and the kidneys [42]. quantification, showing that GQDs do not stay in the kidney but pass through it and are released into the urine, showing a 5 Conclusions fast clearance. An alternative route for the preparation of GQDs was explored The next step was to deepen into the mechanism by which obtaining high yields and the cytotoxicity of the so-obtained GQDs pass through the ultrafiltration barrier of podocytes. materials, presenting an average diameter of 2.9 nm, was The use of specific inhibitors is useful to understand the studied demonstrating their better behavior with respect to preferential NPs internalization way. Previous studies conducted GO. Moreover, in vivo biodistribution experiments were on cancer cells of different tissue origin described GQDs performed, confirming a fast and efficient renal clearance. The caveolae-mediated endocytosis in gastric cancer cells and in GQDs were observed to pass through the glomerular cells breast cancer cells, autophagy in lung cancer and in without any renal toxicity. The mechanism of internalization adenocarcinomic human alveolar basal epithelial cells [36, 40]. was explored and a caveolae-mediated internalization was Moreover, GQDs have been found to enter by endocytosis found in lipid rafts, where ultrafiltration takes place. This and subsequently localized within the lysosome, in neurons, allows bypassing lysosomal degradation. However, future and finally to enter by receptor-mediated endocytosis when multi-dose experiments have to be performed to validate their conjugated with folic acid [36]. This wide variety of internalization biocompatibility, assuming that multiple doses should be modalities and the absence of studies on renal glomerular cells necessary to obtain pharmacological effects. have necessarily led us to clarify which is the entry way into Thanks to these evidences, the obtained GQDs result to be the podocytes. The investigations, obtained by selectively really appealing as possible drug-vector to kidney, being a very blocking different entry pathways, support the hypothesis that safe material for this compartment. So, it is possible to envision in these cells the GQDs use, at least in part, an energy- the use of ultra-small GQDs for drug delivery to ultrafiltration dependent mechanism of endocytosis, as demonstrated by sites, rich in molecules responsible for cell signaling and crucial the weakening of the fluorescence after the addition of for cytoskeletal repair, thus participating in the dynamics sodium azide. After observing the absence of chlorpromazine involved in the treatment of glomerulopathies. effect and the relatively few consequences of wortmannin, clathrin-mediated endocytosis can be excluded. In contrast, the decrease in fluorescence intensity following treatment with Acknowledgements genistein suggests caveolae-mediated endocytosis or clathrin J. M. G.-D. acknowledges Spanish Ministry of Science, Innovation and caveolae-independent mechanisms. The remarkable decrease and Universities for his Juan de la Cierva Incorporación of fluorescence after filipin III definitively indicates that research contract (No. IJCI-2016-27789). This project has caveolae-mediated endocytosis may be the preferential way. received funding from the European Union’s Horizon 2020 To assess the biosafety of GQDs in vivo single-dosing Research and Innovation Programme under the Marie www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 682 Nano Res. 2021, 14(3): 674–683 Skłodowska-Curie Grant Agreement No. 734834 (INFUSION) and graphene quantum dots for optoelectronic and energy devices: and No. 734381 (CARBO-IMmap), and from MIUR. ICN2 A review. Adv. Funct. Mater. 2015, 25, 4929–4947. is supported by the Severo Ochoa program from Spanish [13] Wang, Y.; Li, Z. H.; Li, J. H.; Lin, Y. H. Graphene and graphene MINECO (No. SEV-2017-0706). oxide: Biofunctionalization and applications in biotechnology. Trends Biotechnol. 2011, 29, 205–212. Funding note : Open Access funding provided by Università [14] Wang, S. J.; Cole, I. S.; Li, Q. The toxicity of graphene quantum degli Studi di Trieste within the CRUICARE Agreement. dots. RSC Adv. 2016, 6, 89867–89878. [15] Chong, Y.; Ma, Y. F.; Shen, H.; Tu, X. L.; Zhou, X.; Xu, J. Y.; Dai, J. Electronic Supplementary Material: Supplementary material W.; Fan, S. J.; Zhang, Z. J. The in vitro and in vivo toxicity of (six figures) is available in the online version of this article at graphene quantum dots. Biomaterials 2014, 35, 5041–5048. https://doi.org/10.1007/s12274-020-3096-y. [16] de Menezes, F. D.; dos Reis, S. R. R.; Pinto, S. R.; Portilho, F. L.; Do Vale Chaves e Mello, F.; Helal-Neto, E.; da Silva de Barros, A. Open Access This article is licensed under a Creative Commons O.; Rebêlo Alencar, L. M.; de Menezes, A. 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