Dynamic interactions and intracellular fate of label-free, thin graphene oxide sheets within mammalian cells: role of lateral sheet size

Yingxian Chen; Jack Rivers-Auty; Livia Crica; Katie  Barr; Vinicio Rosano; Adrian Esteban Arranz; Thomas Loret; David Spiller; Cyrill Bussy; Kostas Kostarelos; Sandra Vranic

doi:10.1039/D1NA00133G

Dynamic interactions and intracellular fate of label-free, thin graphene oxide sheets within mammalian cells: role of lateral sheet size

Yingxian Chen, Jack Rivers-Auty, Livia Crica, Katie Barr, Vinicio Rosano, Adrian Esteban Arranz, Thomas Loret, David Spiller, Cyrill Bussy, Kostas Kostarelos, Sandra Vranic

Research output: Contribution to journal › Article › peer-review

Abstract

Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 μm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

Original language	English
Pages (from-to)	4166-4185
Number of pages	20
Journal	Nanoscale Advances
Volume	3
Issue number	14
Early online date	15 Jun 2021
DOIs	https://doi.org/10.1039/D1NA00133G
Publication status	Published - 21 Jul 2021

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CDT in Science and Applications of Graphene and Related Nanomaterial
Grigorieva, I. (PI), Burnett, H. (PGR student), Cusworth, E. (PGR student), Deaconu, D.-A. (PGR student), Dumitriu-Iovanescu, A.-D. (PGR student), Kang, Y.-W. (PGR student), Little, J. (PGR student), Rees, E. (PGR student), Selles, F. (PGR student), Shaker, M. (PGR student), Soong, Y.-C. (PGR student), Swindell, J. (PGR student), Tainton, G. (PGR student), Wood, H. (PGR student), Astles, T. (PGR student), Carl, A. (PGR student), Chen, G. (PGR student), Richard De Latour, H. (PGR student), Dowinton, O. (PGR student), Haskell, S. (PGR student), Hills, K. (PGR student), Hoole, C. (PGR student), Huang, Y. (PGR student), Kalsi, T. (PGR student), Powell, L. (PGR student), Quiligotti, K. (PGR student), Rimmer, J. (PGR student), Smith, L. (PGR student), Thornley, W. (PGR student), Yang, J. (PGR student), Young, W. (PGR student), Zhao, M. (PGR student), Al Busaidi, R. (PGR student), Al Ruqeishi, E. (PGR student), Chadha, A. (PGR student), Chen, M. (PGR student), Dennis, G. (PGR student), Dunn, E. (PGR student), Gamblen, E. (PGR student), Gao, Y. (PGR student), Georgantas, Y. (PGR student), Jiang, Z. (PGR student), Karakasidi, A. (PGR student), Mcellistrim, A. (PGR student), Meehan, M. (PGR student), Okwelogu, E. (PGR student), Taylor, M. (PGR student), Wang, W. (PGR student), Xin, B. (PGR student), Castle, C. (PGR student), Clout, P. (PGR student), Dean, S. D. (PGR student), Fordham, A. (PGR student), Griffin, E. (PGR student), Hardwick, T. (PGR student), Hawkins-Pottier, G. (PGR student), Jones, A. (PGR student), Lewthwaite, K. (PGR student), Monteil, S. (PGR student), Moulsdale, C. (PGR student), Mullan, C. (PGR student), Orts Mercadillo, V. (PGR student), Sanderson, D. (PGR student), Skliueva, I. (PGR student), Skuse, C. (PGR student), Steiner, P. (PGR student), Winstanley, B. (PGR student), Barry, D. (PGR student), Brooks, D. (PGR student), Cai, J. (PGR student), Chen, Y. (PGR student), Chen, C. (PGR student), Draude, A. (PGR student), Emmerson, C. (PGR student), Gavriliuc, V. (PGR student), Greaves, M. (PGR student), Higgins, E. (PGR student), Mcmaster, R. (PGR student), Mcnair, R. (PGR student), O'Brien, C. (PGR student), Peasey, A. (PGR student), Pinter, G. (PGR student), Shao, S. (PGR student), Thomas, D. (PGR student), Thomas, D. (PGR student), Tsim, L. T. B. (PGR student), Wengraf, J. (PGR student), Weston, A. (PGR student), Yu, T. (PGR student), De Libero, H. (PGR student), Chan, K. C. (PGR student), Tan, Y. T. (PGR student) & Thomson, T. (CoI)
1/04/14 → 31/10/25
Project: Other

1 Article

Lung recovery from DNA damage induced by graphene oxide is dependent on size, dose and inflammation profile
de Luna, L. A. V., Loret, T., Fordham, A., Arshad, A., Drummond, M., Dodd, A., Lozano, N., Kostarelos, K. & Bussy, C., 21 Sept 2022, In: Particle and Fibre Toxicology. 19, 1, 62.
Research output: Contribution to journal › Article › peer-review

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@article{85043cd6e35e4f489667e63da3ed3978,

title = "Dynamic interactions and intracellular fate of label-free, thin graphene oxide sheets within mammalian cells: role of lateral sheet size",

abstract = "Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 μm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.",

author = "Yingxian Chen and Jack Rivers-Auty and Livia Crica and Katie Barr and Vinicio Rosano and {Esteban Arranz}, Adrian and Thomas Loret and David Spiller and Cyrill Bussy and Kostas Kostarelos and Sandra Vranic",

note = "Funding Information: Authors would like to acknowledge the funding from EU-Horizon 2020-Graphene Flagship Core 1 project (H2020-EU.1.2., Grant agreement ID: 696656) and EU-Horizon 2020-Graphene Flagship Core 2 project (H2020-EU.1.2.3., Grant agreement ID: 785219). Both Y. C. and L. E. C. would like to acknowledge the studentship from the UK Research and Innovation (UKRI) Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training programme (Graphene NOWNANO CDT; EP/L01548X/1). The authors acknowledge the staff of the Faculty of Biology Medicine and Health EM Facility, in particular Dr Aleksandr Mir-onov, for their expertise and assistance, and the Wellcome Trust for equipment grant support to the EM Facility. The University of Manchester Bioimaging and Single Cell Genomics Facility microscopes used in this study were purchased with grants from the Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust, and the University of Manchester Strategic Fund. The authors also wish to thank Dr N. Hodson from the Bio-AFM Facility for assistance and advice regarding the AFM instrumentation. The authors acknowledge the Manchester Collaborative Centre for Inammation Research (MCCIR) as the funding source for the FACSVerse and Fortessa X20 instrument. Authors also acknowledge EPSRC Harwell XPS facility based at Cardiff University for the provision of X-ray photoelectron spectra. Publisher Copyright: {\textcopyright} The Royal Society of Chemistry.",

year = "2021",

month = jul,

day = "21",

doi = "10.1039/D1NA00133G",

language = "English",

volume = "3",

pages = "4166--4185",

journal = "Nanoscale Advances",

publisher = "Royal Society of Chemistry",

number = "14",

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TY - JOUR

T1 - Dynamic interactions and intracellular fate of label-free, thin graphene oxide sheets within mammalian cells

T2 - role of lateral sheet size

AU - Chen, Yingxian

AU - Rivers-Auty, Jack

AU - Crica, Livia

AU - Barr, Katie

AU - Rosano, Vinicio

AU - Esteban Arranz, Adrian

AU - Loret, Thomas

AU - Spiller, David

AU - Bussy, Cyrill

AU - Kostarelos, Kostas

AU - Vranic, Sandra

N1 - Funding Information: Authors would like to acknowledge the funding from EU-Horizon 2020-Graphene Flagship Core 1 project (H2020-EU.1.2., Grant agreement ID: 696656) and EU-Horizon 2020-Graphene Flagship Core 2 project (H2020-EU.1.2.3., Grant agreement ID: 785219). Both Y. C. and L. E. C. would like to acknowledge the studentship from the UK Research and Innovation (UKRI) Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training programme (Graphene NOWNANO CDT; EP/L01548X/1). The authors acknowledge the staff of the Faculty of Biology Medicine and Health EM Facility, in particular Dr Aleksandr Mir-onov, for their expertise and assistance, and the Wellcome Trust for equipment grant support to the EM Facility. The University of Manchester Bioimaging and Single Cell Genomics Facility microscopes used in this study were purchased with grants from the Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust, and the University of Manchester Strategic Fund. The authors also wish to thank Dr N. Hodson from the Bio-AFM Facility for assistance and advice regarding the AFM instrumentation. The authors acknowledge the Manchester Collaborative Centre for Inammation Research (MCCIR) as the funding source for the FACSVerse and Fortessa X20 instrument. Authors also acknowledge EPSRC Harwell XPS facility based at Cardiff University for the provision of X-ray photoelectron spectra. Publisher Copyright: © The Royal Society of Chemistry.

PY - 2021/7/21

Y1 - 2021/7/21

N2 - Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 μm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

AB - Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 μm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

U2 - 10.1039/D1NA00133G

DO - 10.1039/D1NA00133G

M3 - Article

VL - 3

SP - 4166

EP - 4185

JO - Nanoscale Advances

JF - Nanoscale Advances

IS - 14

ER -

Dynamic interactions and intracellular fate of label-free, thin graphene oxide sheets within mammalian cells: role of lateral sheet size

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CDT in Science and Applications of Graphene and Related Nanomaterial

Research output

Lung recovery from DNA damage induced by graphene oxide is dependent on size, dose and inflammation profile

Cite this