Macrophage inflammatory and metabolic responses to graphene-based nanomaterials differing in size and functionalization


The preparation of graphene-based nanomaterials (GBNs) with appropriate stability and biocompatibility is crucial for their use in biomedical applications. In this work, three GBNs differing in size and/or functionalization have been synthetized and characterized, and their in vitro biological effects were compared. Pegylated graphene oxide (GO-PEG, 200-500 nm) and flavin mononucleotide-stabilized pristine graphene with two different sizes (PG-FMN, 200-400 nm and 100-200 nm) were administered to macrophages, chosen as cellular model due to their key role in the processing of foreign materials and the regulation of inflammatory responses. The results showed that cellular uptake of GBNs was mainly influenced by their lateral size, while the inflammatory potential depended also on the type of functionalization. PG-FMN nanomaterials (both sizes) triggered significantly higher nitric oxide (NO) release, together with some intracellular metabolic changes, similar to those induced by the prototypical inflammatory stimulus LPS. NMR metabolomics revealed that macrophages incubated with smaller PG-FMN displayed increased levels of succinate, itaconate, phosphocholine and phosphocreatine, together with decreased creatine content. The latter two variations were also detected in cells incubated with larger PG-FMN nanosheets. On the other hand, GO-PEG induced a decrease in the inflammatory metabolite succinate and a few other changes distinct from those seen in LPS-stimulated macrophages. Assessment of TNF-alpha secretion and macrophage surface markers (CD80 and CD206) further corroborated the low inflammatory potential of GO-PEG. Overall, these findings revealed distinct phenotypic and metabolic responses of macrophages to different GBNs, which inform on their immunomodulatory activity and may contribute to guide their therapeutic applications.



subject category

Biophysics; Chemistry; Materials Science


Cicuendez, M; Fernandes, M; Ayan-Varela, M; Oliveira, H; Feito, MJ; Diez-Orejas, R; Paredes, JI; Villar-Rodil, S; Vila, M; Portoles, MT; Duarte, IF

our authors


This work was developed in the scope of the projects CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, and CESAM, FCT Ref. UID/AMB/50017/2019, financed by national funds through FCT/MCTES, Portugal. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Further acknowledgments are due to Bruker BioSpinGmbH and the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No 739572 - The Discoveries CTR. I.F.D. and H.O. acknowledge FCT/MCTES for research contracts under the Programs 'Investigador FCT' and Stimulus to Scientific Employment (CEECIND/04050/2017), respectively, and M.C. acknowledges the FCT-awarded research grant SFRH/BPD/101468/2014. This research was also supported by the Spanish Ministerio de Economia y Competitividad (MAT2016-75611-R AEI/FEDER, UE). M.A.-V., S.V.-R. and J.I.P. acknowledge financial support from the Spanish Ministerio de Economia y Competitividad (MINECO) and the European Regional Development Fund (ERDF) through project MAT2015-69844-R, and from the Spanish Ministerio de Ciencia, Innovacion y Universidades, the Spanish Agencia Estatal de Investigacion and ERDF, through project RT12018-100832-B-I00. Partial funding by Plan de Ciencia, Tecnologia e hmovacion (PCTI) 2013-2017 del Principado de Asturias and ERDF (project IDI/2018/000233) is also acknowledged. Thanks also to the staff of the Centro de Citometria y Microscopia de Fluorescencia of the Universidad Complutense de Madrid (Spain) and ICTS Centro Nacional de Microscopia Electronica (Spain) for assistance in the flow cytometry and AFM studies, respectively.

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