Nanoscale View Article Online PAPER View Journal | View Issue Correlative radioimaging and mass spectrometry This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. imaging: a powerful combination to study Open Access Article. Published on 20 February 2023. Downloaded on 11/2/2023 5:06:37 PM. Cite this: Nanoscale, 2023, 15, 5510 14 C-graphene oxide in vivo biodistribution† Hélène Cazier,a Carole Malgorn,b Dominique Georgin,c Nathalie Fresneau,c,d Fabrice Beau,b Kostas Kostarelos, e,f,g Cyrill Bussy, e,f Stéphane Campidelli, d Mathieu Pinault,h Martine Mayne-L’Hermite,h Frédéric Taran, c Christophe Junot,a François Fenaille, a Antoine Sallustrau *c and Benoit Colsch*a Research on graphene based nanomaterials has flourished in the last decade due their unique properties and emerging socio-economic impact. In the context of their potential exploitation for biomedical appli- cations, there is a growing need for the development of more efficient imaging techniques to track the fate of these materials. Herein we propose the first correlative imaging approach based on the combi- nation of radioimaging and mass spectrometry imaging for the detection of Graphene Oxide (GO) labelled with carbon-14 in mice. In this study, 14C-graphene oxide nanoribbons were produced from the 14 oxidative opening of C-carbon nanotubes, and were then intensively sonicated to provide nano-size 14 C-GO flakes. After Intravenous administration in mice, 14C-GO distribution was quantified by radioima- ging performed on tissue slices. On the same slices, MS-imaging provided a highly resolved distribution map of the nanomaterial based on the detection of specific radical anionic carbon clusters ranging from Received 2nd December 2022, C2•− to C9•− with a base peak at m/z 72 (12C) and 74 (14C) under negative laser desorption ionization Accepted 18th February 2023 mass spectrometry (LDI-MS) conditions. This proof of concept approach synergizes the strength of each DOI: 10.1039/d2nr06753f technique and could be advantageous in the pre-clinical development of future Graphene-based bio- rsc.li/nanoscale medical applications. Introduction ology development for higher quality synthesis and versatile surface functionalisation of GBNs has also paved the way Since Geim and Novoselov’s Nobel prize winning discovery in towards their medical and biomedical applications.7–9 2004,1 research on Graphene Based Nanomaterials (GBNs) has However, the commercial exploitation of GBN properties steadily expanded over the years.2,3 GBNs have demonstrated depends critically on their biocompatibility and toxicity unique properties that could be exploited in diverse industrial profile. In fact, there is a growing concern about the risk of sectors (e.g. batteries, touch screens, anti-corrosion primers)4,5 GBN materials towards human health.10 Graphene Oxide (GO) and have important societal impacts.6 Progress in method- is recognised as one of the most preferential GBN materials for biomedical applications11–13 due to its distinctive physico- a chemical properties (large surface area, thinness, high conduc- Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies tivity) and ease to be chemically functionalised.14 In such kind pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France b Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies of applications, a direct and long-term interaction of the nano- pour la Santé (DMTS), SiMos, 91191 Gif-sur-Yvette, France material with the human body can be expected, which imply a c Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies careful evaluation of their biodistribution for health and safety pour la Santé (DMTS), SCBM, 91191 Gif-sur-Yvette, France. considerations. Intravenous (i.v.) route is one of the most E-mail: antoine.sallustrau@cea.fr d common routes of administration for these specific appli- Université Paris Saclay, CEA, CNRS, NIMBE, LICSEN, 91191 Gif-sur-Yvette, France e Nanomedicine Lab, Faculty of Biology, Medicine and Health, AV Hill Building, cations. Several studies using i.v. as a mode of administration University of Manchester, Manchester M13 9PL, UK evidenced an extensive accumulation in the liver, the lungs, f National Graphene Institute, University of Manchester, Manchester M13 9PL, UK spleen or in the kidneys over time.15,16 In a recent study, g Catalan Institute of Nanoscience and Nanotechnology (ICN2), UAB Campus reduced GO was also found in the brain which support the Bellaterra, Barcelona 08193, Spain h possibility for such nanomaterials to cross biological bar- Université Paris-Saclay, CEA, CNRS, NIMBE, LEDNA, 91191 Gif-sur-Yvette, France † Electronic supplementary information (ESI) available. See DOI: https://doi.org/ riers.17 The development of new methods for the long-term 10.1039/d2nr06753f detection and absolute quantification of these materials and 5510 | Nanoscale, 2023, 15, 5510–5518 This journal is © The Royal Society of Chemistry 2023 View Article Online Nanoscale Paper more specifically GO, is undoubtedly an important challenge to better understand the possible risks of long-term biopersis- tence of such materials and in order to make them safer-by- design. Raman spectroscopy imaging,18 photoacoustic spec- troscopy19 or intrinsic fluorescence properties have proven to be useful techniques to detect GO in biological samples. However, they present limitations such as slow imaging speed, weak photoluminescence signals, low sensitivity, and do not This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. allow absolute quantification. Recently, Mass Spectrometry Open Access Article. Published on 20 February 2023. Downloaded on 11/2/2023 5:06:37 PM. Imaging (MSI) has emerged as a powerful tool, providing struc- tural information with an excellent specificity and the capacity to map quantitatively multiple molecular classes such as drugs,20,21 lipids,22 peptides,23 proteins,24 as well as metabolites25,26 or carbon-based nanomaterials.27,28 In 2015, Chen et al. reported the use of laser desorption/ionization mass spectrometry (LDI-MS) for the detection, mapping, and quantification of Carbon Nanotubes (CNTs), Carbon Dots (CDTs) and GO in mouse tissue sections using a specific MS signature consisting of anionic carbon clusters.29 Inspired by these results, we recently published an optimised MSI method to assess the biodistribution of GO and rGO in rodent tissues.30 However, one of the main limitation of MSI is its lower sensitivity than radioimaging,31 despite its better spatial resolution and the fact that it allows identification. Fig. 1 Combinaison of radio-imaging and mass spectrometry imaging Radiolabelling methods (using 188Re, 125I, 99mTc, 198Au, techniques for the detection of 14/12C-graphene oxide. 111 In)32–38 combined with highly sensitive radioimaging tech- niques have already been used to track labelled GO in vitro or in vivo. However, these approaches involve a chemical modifi- Experimental cation of the surface of the nanomaterials. This may in return Materials alter the physicochemical properties of the nanoparticle or the labels may be cleaved from the GBN surface during in vivo cir- Organic solvents (Sigma-Aldrich) were used without further culation, hence affecting their reliable localisation and quanti- purification. 14C-benzene (0.5 mL, 4.63 GBq mmol−1, 58 MBq fication. Nevertheless, the direct incorporation of carbon-14 mg−1) was purchased from Quotient Bioresearch (14C) into the backbone of GBNs has proven to be a method of Radiochemicals Ltd (UK) with a radiochemical and chemical choice to circumvent these drawbacks. In 2013, a graphitized purity > 99.9%. The scintillation fluid (Gold Star, Meridian material was labelled with 14C to assess and quantify its Biotechnology Ltd, (UK) was purchased from PerkinElmer uptake and excretion in Daphnia magna.39 A year later, Czarny (USA). The deionized water used in the experiment was pre- et al. labelled CNTs with 14C and demonstrated their transloca- pared by a Millipore Milli-Q ultra-purification system (resis- tion through the air-blood barrier towards distant organs sup- tance > 18.2 MΩ cm). porting human health risk assessment of CNTs.31 Quantitative measurement of radioactivity using 14C performed by Mao Material characterisation et al. revealed the biodistribution and excretion mechanisms Graphene oxide nanoribbons and graphene oxide were ana- of 14C-labelled carbon-based nanoparticles in mice after intra- lysed by transmission electron microscopy (TEM) (Philips CM tracheal instillation.40 Moreover, the degradation of the 14C- 30; CEA Saclay, DEN-LM2E, France and LVEM5 Low-voltage labelled nanoparticles by liver cells was shown by the capture Transmission Electron Microscope, CORDOUAN Technologies, and quantification of 14CO2 breathed out by mice after intrave- Pessac, France) at an accelerating voltage of 5 kV and a magni- nous injection.41 fication from 25 to 200 kV according to samples. Raman Considering the need for new analytical methods to spectra were recorded on a Horiba Jobin–Yvon LabRam ensure a reliable detection, localisation and quantification of ARAMIS equipped with a laser excitation at 532 nm. The GBN in complex biological environments, we propose here chemical composition of the non-radiolabelled graphene oxide an original correlative imaging approach consisting in com- nanoribbons and graphene oxide was evaluated by XPS (X-ray bining the strengths of both MSI and radio-imaging to induced photoelectron spectroscopy) analysis using a Kratos provide absolute quantification, spatial resolution and Analytical Axis Ultra DLD spectrometer (UK) with monochro- specific signature information, for the in vivo tracking of GO matic Al Kα X-ray radiation (hν = 1486.6 eV). Graphene oxide in mice (Fig. 1). was imaged by Atomic Force Microscopy (AFM) (Multimode 8 This journal is © The Royal Society of Chemistry 2023 Nanoscale, 2023, 15, 5510–5518 | 5511 View Article Online Paper Nanoscale atomic force microscope, Bruker, UK) in tapping mode using ted in four 25 mL tubes. After centrifugation at 25 000 rpm for OTESPA tips (Bruker, UK) with a curvature radius of 10 nm, 15 min (Beckman Coulter, rotor F630), the supernatant was resonant frequency of 300 kHz, and a spring constant of 42 N removed and 20 mL of MilliQ water was added in each tube. A m−1. A high-performance autoradiography imager (β-imager second centrifugation step (25 000 rpm, 15 min) was per- TM 2000, Biospace Lab, Paris, France) was used for µ-particle formed before the supernatant was removed and the pellet dis- counting and absolute radioactivity quantification (detection persed in water (30 mL). After 5 min stirring, the dispersion threshold of 0.01 cpm mm−2 for 14C) allowing real-time radio- was sonicated for 10 min. Then HCl (4 mL, 20%) was active imaging. Mass Spectrometry Imaging experiments added. After 5 min stirring, the solution was centrifuged were performed on an UltraFleXtreme™ Matrix-assisted (25 000 rpm, 15 min). Four additional steps of centrifugation This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Laser Desorption Ionization – tandem Time-of-Flight mass were performed for which the supernatant was systematically Open Access Article. Published on 20 February 2023. Downloaded on 11/2/2023 5:06:37 PM. spectrometer (MALDI-TOF/TOF, Bruker Daltonics, Bremen, removed and replaced by 20 mL of Milli-Q water. After the last Germany). centrifugation step, the pellet was collected and dispersed in Milli-Q water (12 mL). The recovered nanoribbons (17.5 mg, Graphene oxide synthesis 1.46 mg mL−1) were characterized before the synthesis of GO Graphene oxide was produced in a multistep procedure (Fig. 2A) (ESI†). 12 from Carbon Nanotubes synthetized by a catalytic chemical C-GO preparation. To a 10 mL sample of the above GO- vapour deposition process specifically developed for the use of nanoribbons (1.46 mg mL−1) were added 3.5 mL of an benzene as source of carbon, as previously reported.31 aqueous solution of sodium dodecyl benzene sulfonate (SDBS) 12 C-GO nanoribbons preparation. The CNT (21 mg, (2 CMC, 2.2 mg mL−1). The solution was then sonicated 1.75 mmol of carbon) covering the internal surface of the CVD using a Misonix Ultrasonic Convertor (Power 18, 10–11 Watts) reactor were recovered and treated with H2SO4 (95%, 4.4 mL) for 12 hours to obtain a final suspension of GO (13.5 mL, for 60 min at room temperature in a round bottom flask. 1.08 mg mL−1) in deionized water containing SDBS (0.57 mg Then, KMnO4 (228 mg, 1.442 mmol/C) was added and the mL−1). 14 mixture was stirred for 2 hours at 65 °C. The mixture was C-GO nanoribbons and 14C-GO preparation. Starting from cooled to room temperature and poured into a mixture of 20 mg of 14C-CNT (6.438 MBq mg−1), an identical protocol H2O2/ice-cold water (1.6 mL/50 mL). The mixture was distribu- (CNT > GO-Nanoribbons > GO) was followed, providing 3.4 mg Fig. 2 (A) Strategy for the synthesis of 14C-GO; (B) TEM image of 12C-GO; (C) Raman spectrum of 12C-GO; (D) LDI mass spectrum of 12C-GO; (E) AFM heigh image of 14C-GO (scale bar 1 µm); (F) size and thickness distribution of 14C-GO from the AFM heigh image. (G) and (E) TEM image of 14 C-GO; (H) LDI mass spectrum of 12/14C-GO. 5512 | Nanoscale, 2023, 15, 5510–5518 This journal is © The Royal Society of Chemistry 2023 View Article Online Nanoscale Paper of 14C-GO (22 MBq, 6.438 MBq mg−1) in 13.5 mL deionized for fixation. Sections were then rehydrated by successive 1 min water containing SDBS. immersion in 100%, 90% and 70% ethanol and a final 1 min 14/12 C-GO dispersion preparation. An isotopically diluted dis- in water. Tissue sections were then dipped into Haematoxylin persion was used for mice injections and is referred as solution for 30 s, followed by 2 min in water and 30 s in Eosin 14/12 C-GO dispersion in the following text. The 14/12C-GO dis- at 0.5% in water. Tissue sections were then dehydrated by suc- persion was prepared as an equimolar ratio of 14C-GO and cessive immersion in 70%, 90% and 100% ethanol for 1 min. 12 C-GO dispersions: 1 mL of 14C-GO suspension (6.438 MBq Finally, sections were “clarified” in xylene for 2 min. After H&E mg−1; 251 µg mL−1) was added to 1 mL of 12C-GO suspension staining, sections were mounted with ProLong™ mounting (250 µg mL−1) leading to the final 14/12C-GO dispersion (3.219 medium (Fischer Scientific) and a coverslip. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. MBq mg−1; 500 µg mL−1; 1.6 MBq mL−1). Open Access Article. Published on 20 February 2023. Downloaded on 11/2/2023 5:06:37 PM. Radioimaging In vivo experiments A high-performance autoradiography imager was used for the Animals. Mice (Laboratoires Charles River, l’Arbresle, quantitative determination of radioactivity in dried tissue sec- France) were maintained in accordance with French and tions. Correct calibration was verified between each sample European regulations on care and protection of laboratory analysis by counting the radioactivity of a reference sample animals (Directive 2010/63/EU of the European Parliament) (blood with a known quantity of 14C-GO). Organs from and with the agreement of the ethics committee (CEEA) animals exposed to 14/12C-GO were processed and analysed as awarded to Dr V. Dive and agreement APAFIS#4111- described in Table S2.† 2016021613253432v3 from the ministry of national education, higher education and research (France). Pathogen-free, 6-week- Mass spectrometry imaging old, female Balb/c mice weighing on average 20 g were indivi- Methods for 14C-GO and 12C-GO detection were developed on dually housed in polycarbonate cages in a conventional animal an UltraFleXtreme™ MALDI-TOF/TOF mass spectrometer as facility and had access to food and drink ad libitum. described before.30 The MALDI-TOF instrument operated in Material exposure and tissue harvesting. Three mice were the negative ionization reflectron mode, using an optimized exposed to 50 and 75 μg of 14/12C-GO dispersion by intravenous extraction delay set at 10 ns. MS analyses were first performed (tail vein) injection. In parallel, one mouse was exposed to the at a 2 kHz laser repetition rate with a Smartbeam II laser vehicle alone (SDBS) as negative control. Mice were sacrificed ((Bruker Daltonics, Germany), which delivered a gross flat top 15 minutes after exposure to 14/12C-GO by intraperitoneal injec- beam profile with small laser beam focusing at 90% of tion of Pentobarbital (Exagon®, Pentobarbital, Gedeon fluence. Source and TOF pressure were below 1 × 10−7 mbar Richter, Austria). All organs were harvested and immediately and voltages were adjusted as follows: IS1 = 20.02 kV, IS2 = snap-frozen at −80 °C by immersion in a mixture of dry ice 18.02 kV, lens = 6.99 kV, reflectron = 21.03 kV, and reflectron and isopentane to prevent redistribution of the 14/12C-GO. 2 = 10.93 kV. MS measurements were performed from m/z 0 to Tissue sections. Snap-frozen lungs, liver, kidneys and spleen 200 with 1000 accumulated shots per spectrum. Images were of exposed and control mice were sectioned at 12 µm thickness acquired with a spatial resolution set at 25 µm for biodistribu- using a cryostat (LEICA Microsystems, France) in which the tion studies and 100 µm for quantification assay. Data proces- chamber temperature (Tchamb) was adjusted to −18 °C and the sing was achieved using FlexImaging 4.0 or SCiLS Lab 2023a object temperature (Tobj ) to −20 °C. The cryotome was carefully Pro (Bruker Daltonics, Bremen, Germany). The molecular cleaned by a water/ethanol solution between the processing of images were reconstituted based on the average intensities of each sample to remove any trace of radioactivity and to avoid the C6•− anionic radical ion at m/z 72, which proved to be the cross-contamination between different organs. For each organ, most intense radical anion for the 14C-GO signature.30 some sections were mounted onto Indium tin oxide coated glass slides (ITO) specific for MSI analyses, while adjacent sec- tions were mounted on Superfrost plus® gold slides (Thermo Results and discussion Scientific) to perform Hematoxylin and Eosin (H&E) staining. Adhesive aluminium foil (3M, Diegem, Belgium) was added on Preparation and characterisation of graphene oxide the non-conductive side of ITO slides. The latter were found to First developed and optimized with carbon-12 materials, the be compatible for both MSI and Radioimaging techniques following strategy was applied to synthesize 14C-GO. It started thanks to the conductive adhesive foil addition on the non- with the longitudinal opening or “unzipping” of carbon nano- conductive side. Moreover, the above mentioned adhesive alu- tubes synthesized by Catalytic Chemical Vapor Deposition minium foil is commonly used to allow radio-imaging acqui- (CCVD)42–44 from 14C-benzene, as previously reported.31 The 14 sition. Tissue sections mounted on ITO slide were maintained C-CNT were then oxidized in presence of KMnO4 and H2SO4 at room temperature for one day in the presence of silica gel leading to the formation of 14C-graphene oxide nanoribbons. pellets to ensure complete drying before β-imager acquisition. After being washed and centrifuged several times, the nano- Tissue staining. H&E staining was carried out using solu- ribbons were sonicated for 12 hours using a Misonix tions whose compositions are described in Table S1.† First, Ultrasonic Convertor in an aqueous solution of SDBS leading tissues were immersed for 2 min in 10% formalin in ethanol to a suspension of 14C-GO (3.4 mg, 22 MBq, 6.438 MBq mg−1). This journal is © The Royal Society of Chemistry 2023 Nanoscale, 2023, 15, 5510–5518 | 5513 View Article Online Paper Nanoscale As expected, the specific activity of 14C-GO measured by An isotopically diluted graphene oxide dispersion of 14C-GO β-imager remained identical to the starting CNTs (6.438 MBq and 12C-GO in SDBS referred as 14/12C-GO dispersion (3.2 MBq mg−1). mg−1, 500 µg mL−1, 1.6 MBq mL−1) was prepared in order to The physicochemical characterization of 12C-GO and increase the MSI signal of 14C-GO and avoid saturation of the 14 C-GO was performed to confirm the consistency of the pro- radio-imaging signal. Mice were then exposed to 50 µg (161 duction method and quality of the engineered materials. kBq) and 75 µg (241 kBq) corresponding respectively to the Raman analysis performed on 12C-GO showed peaks at injection of 100 µl and 150 µl of the 14/12C-GO dispersion by 1363 cm−1 (D band), 1611 cm−1 (G band) and at 2730 cm−1 intravenous administration. Mice were sacrificed 15 min after (2D band), all of which are characteristic of oxidized graphenic injection. Lungs, liver, spleen and kidneys were collected, This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. materials. The D band stands for the presence of defects (Csp3 snap-frozen, cryo-sectioned and mounted on either ITO or Open Access Article. Published on 20 February 2023. Downloaded on 11/2/2023 5:06:37 PM. and structural defects) in the sp2 structure and the G band Superfrost® slides for further analysis. corresponds to the remaining the graphitic mode of graphene. The defects in the structure were evaluated by the ID/IG ratio that equals to ∼1 here. Transmission electron microscopy (TEM) analysis of 12C-GO showed nanometre scale graphene oxide particles (<100 nm) (Fig. S8†) with a size distribution similar to 14C-GO and a narrow distribution under 100 nm (Fig. 2) corresponding to 75% of all particles. The morphology of 14C-GO was determined by AFM. It showed that the lateral dimension ranged from 50 to 400 nm, with a main size distri- bution centred below 100 nm, in agreement with TEM imaging. The average thickness was about 1 nm, indicating that 14C-GO nanoparticles were mainly made of 1 to 2 layers of graphene oxide sheets, given that GO interlayer distance is 0.86 nm.45 XPS analyses were performed only on 12C-GO nano- ribbons and 12C-GO to avoid any radioactive contamination of the equipment. The deconvolution C1s spectrum of 12C-GO nanoribbons exhibits 3 major peaks at 284.5, 287, and 288 eV. The peak at 284.5 eV is assigned to C–C and CvC. The peak at 287 eV is assigned to C–O species (C-OH and epoxy groups), and the remaining shoulder at 288 eV corresponds to the CvO binding energy. A similar peak distribution was observed for 12 C-GO. However the contribution of the C–C/CvC peak and the C–O peak changes from the GO nanoribbons to GO (Fig. S3 and S11†) with a decrease of the C–O peak. However, there is no major change observed in the CvO species. Additionally, XPS of 12C-GO nanoribbons indicates that the carbon content was the dominant feature (ca. 67.5%) as expected, and the oxygen content was found to reach almost a third of the total amount (ca. 31.5%), confirming the oxidized nature of the nanoribbons (Fig. S3 and S4†). Quantitative ana- lysis of 12C-GO also indicated that carbon was predominant (ca. 69.0%) with a significant proportion of oxygen (ca. 30.3%) (Fig. S11 and S12†). Furthermore, the C/O ratio increases from 2.14 in GO nano- ribbons to 2.28 in GO, revealing the elimination of some oxygen functional groups after the ultrasonication step. This was confirmed by the work of Le et al.46 where they described Fig. 3 MSI analysis of a lung section from a mouse exposed to 50 µg of the effect of sonication on GO leading to the removal of 14 C/12C-GO with a spatial resolution of 25 µm. The MS image was various oxygen groups and leading to increased C/O ratios. acquired with an accumulation of 1000 shots per spectrum and per pixel at 2 kHz with a DIE of 10 ns, small laser focus and 90% laser fluency. (A) Molecular image of 12C-GO was represented using m/z 72 Combining radio and MS imaging ion. (B) H&E staining of the same lung section. (C) MS profile of 14/ 12 C-GO. (D) MS images of the m/z 74 corresponding to 14C-GO in the Before assessing the biodistribution of GO in tissues by MSI same section. (E) Overlay ( purple) of the two MS images, namely the and radio-imaging, adequate sample preparation and experi- m/z 72 (blue) and the m/z 74 (red), corresponding to the 14/12C-GO mental conditions have been optimized as mentioned below. (14C/12C6•−). 5514 | Nanoscale, 2023, 15, 5510–5518 This journal is © The Royal Society of Chemistry 2023 View Article Online Nanoscale Paper To perform optimal GO detection in tissue sections by MSI, lung section, namely the m/z 72 (blue; Fig. 3A) and the m/z 74 the methodology previously developed for the detection of (red; Fig. 3D), together with the corresponding H&E staining of unlabelled GO and rGO30 was used for the radiolabelled GO. an adjacent lung section (Fig. 3B) revealed an even distribution In this case, ITO slides have been used to provide both radioi- of 14/12C-GO materials across the lungs (Fig. 3E). maging and MSI analysis of the same tissue slice. LDI-MS After 14/12C-GO sample preparation and the validation of a parameters were thus optimised directly on the 14/12C-GO specific LDI-MS signal for 14/12C-GO, radio-imaging and MSI exposed tissues. For that, an extraction delay of 10 nano- were successively performed on the same slice to investigate 14/12 seconds, a fluence of 90% in reflectron acquisition and in C-GO fate in vivo. Here, ITO slides used specifically for negative ionisation mode were used. In respect to the MSI MSI analyses, was also tested and validated for radioimaging This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. laser, the parameters were fixed with the “minimum” focus, a acquisition allowing both radioimaging and MSI analysis of Open Access Article. Published on 20 February 2023. Downloaded on 11/2/2023 5:06:37 PM. random movement of the laser within the “raster”, and a the same tissue slice. Since MSI is a destructive analytical tech- spatial resolution of either 100 µm or 25 μm for biodistribu- nique, radioimaging analysis was thus performed first provid- tion studies. Finally, an internal calibration was performed on ing a sensitive assessment of the GO biodistribution and the expected theoretical signature before acquisition of the MS allowing to select the most relevant organs or area onto which images in order to guarantee mass measurement accuracy. MSI will be carried out. Following these first steps, we then investigated the LDI-MS β-Imager analysis has been performed based on the average signature of 14C-GO and ability to produce specific MSI images measurements of three adjacent organ sections. The GO (Fig. 3). To do so, a comparative analysis of 14C-GO and 12C-GO amount found per section was then normalised to the total was carried out on lung tissues exposed to 14/12C-GO. LDI-MS organ weight and expressed in percentages. Cumulating the profiles of 12C-GO (Fig. 2D) and 14/12C-GO (Fig. 3C) indicated four organs analysed, a total of 96.8% and 97.1% of the total similar radical anionic carbon clusters ranging from C2•− to injected doses was recovered for the 50 µg and 75 µg doses, C9•−, which was consistent with our previous results.27 The respectively (Table S3†). Biodistribution images (Fig. 4B and most intense peak at m/z 71.99 was detected for both labelled Fig. S18†) show the predominant accumulation of the labelled and unlabelled nanoparticles. Interestingly, a slightly more GO in the liver for both injected doses (i.e., 50 and 75 µg) (n = intense peak was observed at m/z 74, related to 14C, due to the 3 slices from the same mouse) corresponding respectively to incorporation of the carbon isotope (one 14C for 35 12C) into 51% and 57% of the total injected dose. A strong signal was the structure of GO. The overlay of the two MS images of a also found in lungs with 45% and 39% for 50 µg and 75 µg Fig. 4 Comparison between H&E, radio and MS imaging of lung sections from mice exposed to 50 µg and 75 µg of 14/12C-GO. (A) H&E staining of a successive slice of lung section. (B) β-Imager acquisition of 50 µg and 75 µg injection dose with a spatial resolution of 150 µm. (C) MSI analysis of the same lung section from mice exposed to 50 µg and 75 µg of 14/12C-GO with a spatial resolution of 25 (insert C.1 and C.2) and 100 µm. The MS images were acquired with an accumulation of 1000 shots per spectrum and per pixel at 2 kHz with an extraction delay of 10 ns, small laser focus and 90% laser fluency. Molecular images of GO were represented using the overlay of maps ( purple) obtained for m/z 72 (blue) and 74 (red) ions. This journal is © The Royal Society of Chemistry 2023 Nanoscale, 2023, 15, 5510–5518 | 5515 View Article Online Paper Nanoscale injected doses. Substantial accumulation in lungs is consistent Conclusions with previous short-term distribution studies after GO intrave- nous administration reported in the literature.47,48 Following In the present study, we demonstrated how the combination of iv injection, GO particles are large enough to be entrapped in radioimaging and MS imaging can be used as a correlative the first capillary networks they encountered (i.e., lungs and imaging approach to provide absolute quantification and high liver), which in turn reduces their transport and accumulation resolution spatial distribution of 14C-labelled graphene oxide in other organs. Indeed, much less radioactivity was found in in selected organs of accumulation after intravenous adminis- the spleen and kidneys where less than 1% of the total injected tration in mice. dose was detected, irrespective of the injected dose. Despite Labelled GO nanoparticles dispersed in 0.05% SDBS in This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. this quantitative assessment of the radioactivity distribution in water, were detectable by LDI-MS without addition of MALDI Open Access Article. Published on 20 February 2023. Downloaded on 11/2/2023 5:06:37 PM. the different organs, the low spatial resolution of β-imaging matrix, owing to GO aromatic structure favouring desorption/ prevented a more precise evaluation of the GO distribution in ionisation phenomena. Moreover, a profile of carbon and the different organs of interest. MSI was therefore used as a radical clusters was detected with structures from C2•− to C9•−. complementary technique providing higher resolution to cir- MS imaging parameters were then optimised to enable the cumvent this drawback. MSI was thus performed on liver and successful detection of GO signature in tissue sections. lungs for both injected doses as they correspond to the main Biodistribution studies with both β-imager and MSI were targeted organs. finally performed on the same tissue section for each organ Since MSI is mainly used for molecular mapping, the acqui- tested. Quantitative assessment performed with β-imager sition of mass spectra at different coordinates allows the repre- showed greater amounts of GO in the liver than in the lungs, sentation of several molecules or nanoparticles with an spleen and kidneys. Biodistribution within each organ was average spatial resolution of 20 μm or less.49,50 This spatial obtained with a higher spatial resolution for MSI (25 μm) than resolution corresponds to the distance between two acqui- for β-imager (150 μm) even though the best signal was obtain sitions on the same tissue slice. MSI was first performed with for the lungs. a spatial resolution of 100 µm providing a preliminary assess- Combining the strength of radioimaging quantification and ment of the distribution of GO in the different organs. While MS imaging spatial resolution, the present work highlights the sensitivity of MSI did not allow the detection of the lower benefit of the proposed bimodal approach to provide simul- amount of labelled GO present in the spleen and kidneys due taneously absolute quantification and high resolution biodis- to the estimated limit of detection (LOD) of our LDI-MSI tribution of GBN accumulated within biological tissues. method (above 5 ng mg−1 of GO),30 14/12C-GO distribution in Application of this proof of concept based on advanced quanti- both lungs and liver was spatially resolved by this method fication and analytical methods for the detection and quantifi- (Fig. S19 and S20†). However, MSI images of the liver sections cation of GO could be extended to other GBN for the safe and showed fewer GO specific signals compared to the lungs, sustainable development of future GBN-based industrial bio- which might be counterintuitive considering the distribution medical applications. results provided by radioimaging. This can be explained by the physiological differences between liver and lungs, and by the fact that MSI images only consider the biodistribution of GO Author contributions on the slice surface of each organ, while the radioimaging quantification is calculated according to the weight of the The manuscript was written through contributions of all whole organ. MSI was thus performed on lungs with a spatial authors. All authors have given approval to the final version of resolution of 25 µm (Fig. 4C and Fig. S21†) providing higher the manuscript. spatial resolution than β-imager, which has an average resolu- tion of 150 µm (Fig. 4B).51 We decided then to compare radioimaging and MSI on Ethical statements lung tissue images. Here, 14/12C-GO images of lungs obtained by LDI-MSI overlapped with radioactivity areas revealed by All animal procedures were performed in accordance for Care β-imaging and thus confirmed the presence of labelled GO and Use of Laboratory Animals of the Directive 2010/63/EU of nanoparticles in exposed mice (Fig. 4). Furthermore, MSI pro- the European Parliament, which had been approved by the vided additional information on the biodistribution of Ministry of Agriculture, France. The project was approved by 14/12 C-GO in tissues by highlighting the alveolar aspect of lung the French Ethics Committee CEEA (Comité d’Ethique en tissue (Fig. 4C1 and C2). Areas without radioactivity were also Expérimentation Animale). The French Ethics Committee CEEA found in MSI images with a finer resolution, allowing an accu- (Comité d’Ethique en Expérimentation Animale) is responsible rate superposition between MSI, β-imaging and H&E staining for overseeing the provisions for the care and well-being of images. Interestingly, due to the spatial resolution gap animals used for research and educational purposes at between MSI and β-imaging, radio-imaging seems to show a CEA-Saclay and serves the public by ensuring compliance with decreasing intensity of the 14/12C-GO from the centre to the all legal and ethical standards regarding the use of vertebrate edges of the section; this was not found in MSI. animals in research and teaching at CEA-Saclay. 5516 | Nanoscale, 2023, 15, 5510–5518 This journal is © The Royal Society of Chemistry 2023 View Article Online Nanoscale Paper Conflicts of interest 16 T. Fan, L. Yan, S. He, Q. Hong, F. Ai, S. He, T. 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