Publications
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Banning carbon nanotubes would be scientifically unjustified and damaging to innovation
Heller D.A., Jena P.V., Pasquali M., Kostarelos K., Delogu L.G., Meidl R.E., Rotkin S.V., Scheinberg D.A., Schwartz R.E., Terrones M., Wang Y.H., Bianco A., Boghossian A.A., Cambré S., Cognet L., Corrie S.R., Demokritou P., Giordani S., Hertel T., Ignatova T., Islam M.F., Iverson N.M., Jagota A., Janas D., Kono J., Kruss S., Landry M.P., Li Y., Martel R., Maruyama S., Naumov A.V., Prato M., Quinn S.J., Roxbury D., Strano M.S., Tour J.M., Weisman R.B., Wenseleers W., Yudasaka M. Nature Nanotechnology; 15 (3): 164 - 166. 2020. 10.1038/s41565-020-0656-y. IF: 31.538
[No abstract available]
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Graphene, other carbon nanomaterials and the immune system: toward nanoimmunity-by-design
Arianna Gazzi, Laura Fusco, Marco Orecchioni, Silvia Ferrari, Giulia Franzoni, J Stephen Yan, Matthias Rieckher, Guotao Peng, Matteo Andrea Lucherelli, Isabella Anna Vacchi, Ngoc Do Quyen Chau, Alejandro Criado, Akcan Istif, Donato Mancino, Antonio Dominguez, Hagen Eckert, Ester Vázquez, Tatiana Da Ros, Paola Nicolussi, Vincenzo Palermo, Björn Schumacher, Gianaurelio Cuniberti, Yiyong Mai, Cecilia Clementi, Matteo Pasquali, Xinliang Feng, Kostas Kostarelos, Acelya Yilmazer, Davide BedognettI, Bengt Fadeel, Maurizio Prato, Alberto Bianco and Lucia Gemma Delogu Journal of Physics: Materials; 3 (34009) 2020. 10.1088/2515-7639/ab9317. IF: 0.000
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Grouping all carbon nanotubes into a single substance category is scientifically unjustified
Fadeel B., Kostarelos K. Nature Nanotechnology; 15 (3): 164. 2020. 10.1038/s41565-020-0654-0. IF: 31.538
[No abstract available]
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Intracerebral Injection of Graphene Oxide Nanosheets Mitigates Microglial Activation Without Inducing Acute Neurotoxicity: A Pilot Comparison to Other Nanomaterials
Portioli C., Bussy C., Mazza M., Lozano N., Jasim D.A., Prato M., Bianco A., Bentivoglio M., Kostarelos K. Small; 16 (48, 2004029) 2020. 10.1002/smll.202004029. IF: 11.459
Carbon-based nanomaterials (CNMs) are being explored for neurological applications. However, systematic in vivo studies investigating the effects of CNM nanocarriers in the brain and how brain cells respond to such nanomaterials are scarce. To address this, functionalized multiwalled carbon nanotubes and graphene oxide (GO) sheets are injected in mice brain and compared with charged liposomes. The induction of acute neuroinflammatory and neurotoxic effects locally and in brain structures distant from the injection site are assessed up to 1 week postadministration. While significant neuronal cell loss and sustained microglial cell activation are observed after injection of cationic liposomes, none of the tested CNMs induces either neurodegeneration or microglial activation. Among the candidate nanocarriers tested, GO sheets appear to elicit the least deleterious neuroinflammatory profile. At molecular level, GO induces moderate activation of proinflammatory markers compared to vehicle control. At histological level, brain response to GO is lower than after vehicle control injection, suggesting some capacity for GO to reduce the impact of stereotactic injection on brain. While these findings are encouraging and valuable in the selection and design of nanomaterial-based brain delivery systems, they warrant further investigations to better understand the mechanisms underlying GO immunomodulatory properties in brain. © 2020 Wiley-VCH GmbH
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Multiparametric Profiling of Engineered Nanomaterials: Unmasking the Surface Coating Effect
Gallud A., Delaval M., Kinaret P., Marwah V.S., Fortino V., Ytterberg J., Zubarev R., Skoog T., Kere J., Correia M., Loeschner K., Al-Ahmady Z., Kostarelos K., Ruiz J., Astruc D., Monopoli M., Handy R., Moya S., Savolainen K., Alenius H., Greco D., Fadeel B. Advanced Science; 7 (22, 2002221) 2020. 10.1002/advs.202002221. IF: 15.840
Despite considerable efforts, the properties that drive the cytotoxicity of engineered nanomaterials (ENMs) remain poorly understood. Here, the authors inverstigate a panel of 31 ENMs with different core chemistries and a variety of surface modifications using conventional in vitro assays coupled with omics-based approaches. Cytotoxicity screening and multiplex-based cytokine profiling reveals a good concordance between primary human monocyte-derived macrophages and the human monocyte-like cell line THP-1. Proteomics analysis following a low-dose exposure of cells suggests a nonspecific stress response to ENMs, while microarray-based profiling reveals significant changes in gene expression as a function of both surface modification and core chemistry. Pathway analysis highlights that the ENMs with cationic surfaces that are shown to elicit cytotoxicity downregulated DNA replication and cell cycle responses, while inflammatory responses are upregulated. These findings are validated using cell-based assays. Notably, certain small, PEGylated ENMs are found to be noncytotoxic yet they induce transcriptional responses reminiscent of viruses. In sum, using a multiparametric approach, it is shown that surface chemistry is a key determinant of cellular responses to ENMs. The data also reveal that cytotoxicity, determined by conventional in vitro assays, does not necessarily correlate with transcriptional effects of ENMs. © 2020 The Authors. Published by Wiley-VCH GmbH
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Nano-scavengers for blood biomarker discovery in ovarian carcinoma
Hadjidemetriou M., Papafilippou L., Unwin R.D., Rogan J., Clamp A., Kostarelos K. Nano Today; 34 (100901) 2020. 10.1016/j.nantod.2020.100901. IF: 16.907
The development and implementation of biomarker-based screening tools for ovarian cancer require novel analytical platforms to enable the discovery of biomarker panels that will overcome the limitations associated with the clinically used CA-125.The systematic discovery of protein biomarkers directly from human plasma using proteomics remains extremely challenging, due to the wide concentration range of plasma proteins. Here, we describe the use of lipid-based nanoparticles (NPs) as an ‘omics’ enrichment tool to amplify cancer signals in the blood and to uncover disease specific signatures. We aimed to exploit the spontaneous interaction of clinically-used liposomes (Caelyx®) with plasma proteins, also known as’ protein corona’ formation, in order to facilitate the discovery of previously unreported differentially abundant molecules. Caelyx® liposomes were incubated with plasma samples obtained from advanced ovarian carcinoma patients and healthy donors and corona-coated liposomes were subsequently recovered. Comprehensive comparison between ‘healthy’ and ‘diseased’ corona samples by label-free proteomics resulted in the identification of multiple differentially abundant proteins. Moreover, immunoassay-based validation of selected proteins demonstrated the potential of nanoparticle-platform proposed to discover novel molecules with great diagnostic potential. This study proposes a nanoparticle-enabled workflow for plasma proteomic analysis in healthy and diseased states and paves the way for further work needed to discover and validate panels of novel biomarkers for disease diagnosis and monitoring. © 2020
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Nanoscale nights of COVID-19
Kostarelos K. Nature Nanotechnology; 15 (5): 343 - 344. 2020. 10.1038/s41565-020-0687-4. IF: 31.538
[No abstract available]
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Nitric oxide-dependent biodegradation of graphene oxide reduces inflammation in the gastrointestinal tract
Peng G., Montenegro M.F., Ntola C.N.M., Vranic S., Kostarelos K., Vogt C., Toprak M.S., Duan T., Leifer K., Bräutigam L., Lundberg J.O., Fadeel B. Nanoscale; 12 (32): 16730 - 16737. 2020. 10.1039/d0nr03675g. IF: 6.895
Understanding the biological fate of graphene-based materials such as graphene oxide (GO) is crucial to assess adverse effects following intentional or inadvertent exposure. Here we provide first evidence of biodegradation of GO in the gastrointestinal tract using zebrafish as a model. Raman mapping was deployed to assess biodegradation. The degradation was blocked upon knockdown of nos2a encoding the inducible nitric oxide synthase (iNOS) or by pharmacological inhibition of NOS using l-NAME, demonstrating that the process was nitric oxide (NO)-dependent. NO-dependent degradation of GO was further confirmed in vitro by combining a superoxide-generating system, xanthine/xanthine oxidase (X/XO), with an NO donor (PAPA NONOate), or by simultaneously producing superoxide and NO by decomposition of SIN-1. Finally, by using the transgenic strain Tg(mpx:eGFP) to visualize the movement of neutrophils, we could show that inhibition of the degradation of GO resulted in increased neutrophil infiltration into the gastrointestinal tract, indicative of inflammation. © 2020 The Royal Society of Chemistry.
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Production and processing of graphene and related materials
Backes C., Abdelkader A.M., Alonso C., Andrieux-Ledier A., Arenal R., Azpeitia J., Balakrishnan N., Banszerus L., Barjon J., Bartali R., Bellani S., Berger C., Berger R., Ortega M.M.B., Bernard C., Beton P.H., Beyer A., Bianco A., Bøggild P., Bonaccorso F., Barin G.B., Botas C., Bueno R.A., Carriazo D., Castellanos-Gomez A., Christian M., Ciesielski A., Ciuk T., Cole M.T., Coleman J., Coletti C., Crema L., Cun H., Dasler D., De Fazio D., Díez N., Drieschner S., Duesberg G.S., Fasel R., Feng X., Fina A., Forti S., Galiotis C., Garberoglio G., García J.M., Garrido J.A., Gibertini M., Gölzhäuser A., Gómez J., Greber T., Hauke F., Hemmi A., Hernandez-Rodriguez I., Hirsch A., Hodge S.A., Huttel Y., Jepsen P.U., Jimenez I., Kaiser U., Kaplas T., Kim H., Kis A., Papagelis K., Kostarelos K., Krajewska A., Lee K., Li C., Lipsanen H., Liscio A., Lohe M.R., Loiseau A., Lombardi L., López M.F., Martin O., Martín C., Martínez L., Martin-Gago J.A., Martínez J.I., Marzari N., Mayoral A., McManus J., Melucci M., Méndez J., Merino C., Merino P., Meyer A.P., Miniussi E., Miseikis V., Mishra N., Morandi V., Munuera C., Muñoz R., Nolan H., Ortolani L., Ott A.K., Palacio I., Palermo V., Parthenios J., Paste 2D Materials; 7 (2, 022001) 2020. 10.1088/2053-1583/ab1e0a. IF: 7.140
We present an overview of the main techniques for production and processing of graphene and related materials (GRMs), as well as the key characterization procedures. We adopt a 'hands-on' approach, providing practical details and procedures as derived from literature as well as from the authors' experience, in order to enable the reader to reproduce the results. Section I is devoted to 'bottom up' approaches, whereby individual constituents are pieced together into more complex structures. We consider graphene nanoribbons (GNRs) produced either by solution processing or by on-surface synthesis in ultra high vacuum (UHV), as well carbon nanomembranes (CNM). Production of a variety of GNRs with tailored band gaps and edge shapes is now possible. CNMs can be tuned in terms of porosity, crystallinity and electronic behaviour. Section II covers 'top down' techniques. These rely on breaking down of a layered precursor, in the graphene case usually natural crystals like graphite or artificially synthesized materials, such as highly oriented pyrolythic graphite, monolayers or few layers (FL) flakes. The main focus of this section is on various exfoliation techniques in a liquid media, either intercalation or liquid phase exfoliation (LPE). The choice of precursor, exfoliation method, medium as well as the control of parameters such as time or temperature are crucial. A definite choice of parameters and conditions yields a particular material with specific properties that makes it more suitable for a targeted application. We cover protocols for the graphitic precursors to graphene oxide (GO). This is an important material for a range of applications in biomedicine, energy storage, nanocomposites, etc. Hummers' and modified Hummers' methods are used to make GO that subsequently can be reduced to obtain reduced graphene oxide (RGO) with a variety of strategies. GO flakes are also employed to prepare three-dimensional (3d) low density structures, such as sponges, foams, hydro- or aerogels. The assembly of flakes into 3d structures can provide improved mechanical properties. Aerogels with a highly open structure, with interconnected hierarchical pores, can enhance the accessibility to the whole surface area, as relevant for a number of applications, such as energy storage. The main recipes to yield graphite intercalation compounds (GICs) are also discussed. GICs are suitable precursors for covalent functionalization of graphene, but can also be used for the synthesis of uncharged graphene in solution. Degradation of the molecules intercalated in GICs can be triggered by high temperature treatment or microwave irradiation, creating a gas pressure surge in graphite and exfoliation. Electrochemical exfoliation by applying a voltage in an electrolyte to a graphite electrode can be tuned by varying precursors, electrolytes and potential. Graphite electrodes can be either negatively or positively intercalated to obtain GICs that are subsequently exfoliated. We also discuss the materials that can be amenable to exfoliation, by employing a theoretical data-mining approach. The exfoliation of LMs usually results in a heterogeneous dispersion of flakes with different lateral size and thickness. This is a critical bottleneck for applications, and hinders the full exploitation of GRMs produced by solution processing. The establishment of procedures to control the morphological properties of exfoliated GRMs, which also need to be industrially scalable, is one of the key needs. Section III deals with the processing of flakes. (Ultra)centrifugation techniques have thus far been the most investigated to sort GRMs following ultrasonication, shear mixing, ball milling, microfluidization, and wet-jet milling. It allows sorting by size and thickness. Inks formulated from GRM dispersions can be printed using a number of processes, from inkjet to screen printing. Each technique has specific rheological requirements, as well as geometrical constraints. The solvent choice is critical, not only for the GRM stability, but also in terms of optimizing printing on different substrates, such as glass, Si, plastic, paper, etc, all with different surface energies. Chemical modifications of such substrates is also a key step. Sections IV-VII are devoted to the growth of GRMs on various substrates and their processing after growth to place them on the surface of choice for specific applications. The substrate for graphene growth is a key determinant of the nature and quality of the resultant film. The lattice mismatch between graphene and substrate influences the resulting crystallinity. Growth on insulators, such as SiO2, typically results in films with small crystallites, whereas growth on the close-packed surfaces of metals yields highly crystalline films. Section IV outlines the growth of graphene on SiC substrates. This satisfies the requirements for electronic applications, with well-defined graphene-substrate interface, low trapped impurities and no need for transfer. It also allows graphene structures and devices to be measured directly on the growth substrate. The flatness of the substrate results in graphene with minimal strain and ripples on large areas, allowing spectroscopies and surface science to be performed. We also discuss the surface engineering by intercalation of the resulting graphene, its integration with Si-wafers and the production of nanostructures with the desired shape, with no need for patterning. Section V deals with chemical vapour deposition (CVD) onto various transition metals and on insulators. Growth on Ni results in graphitized polycrystalline films. While the thickness of these films can be optimized by controlling the deposition parameters, such as the type of hydrocarbon precursor and temperature, it is difficult to attain single layer graphene (SLG) across large areas, owing to the simultaneous nucleation/growth and solution/precipitation mechanisms. The differing characteristics of polycrystalline Ni films facilitate the growth of graphitic layers at different rates, resulting in regions with differing numbers of graphitic layers. High-quality films can be grown on Cu. Cu is available in a variety of shapes and forms, such as foils, bulks, foams, thin films on other materials and powders, making it attractive for industrial production of large area graphene films. The push to use CVD graphene in applications has also triggered a research line for the direct growth on insulators. The quality of the resulting films is lower than possible to date on metals, but enough, in terms of transmittance and resistivity, for many applications as described in section V. Transfer technologies are the focus of section VI. CVD synthesis of graphene on metals and bottom up molecular approaches require SLG to be transferred to the final target substrates. To have technological impact, the advances in production of high-quality large-area CVD graphene must be commensurate with those on transfer and placement on the final substrates. This is a prerequisite for most applications, such as touch panels, anticorrosion coatings, transparent electrodes and gas sensors etc. New strategies have improved the transferred graphene quality, making CVD graphene a feasible option for CMOS foundries. Methods based on complete etching of the metal substrate in suitable etchants, typically iron chloride, ammonium persulfate, or hydrogen chloride although reliable, are time- and resourceconsuming, with damage to graphene and production of metal and etchant residues. Electrochemical delamination in a low-concentration aqueous solution is an alternative. In this case metallic substrates can be reused. Dry transfer is less detrimental for the SLG quality, enabling a deterministic transfer. There is a large range of layered materials (LMs) beyond graphite. Only few of them have been already exfoliated and fully characterized. Section VII deals with the growth of some of these materials. Amongst them, h-BN, transition metal tri- and di-chalcogenides are of paramount importance. The growth of h-BN is at present considered essential for the development of graphene in (opto) electronic applications, as h-BN is ideal as capping layer or substrate. The interesting optical and electronic properties of TMDs also require the development of scalable methods for their production. Large scale growth using chemical/physical vapour deposition or thermal assisted conversion has been thus far limited to a small set, such as h-BN or some TMDs. Heterostructures could also be directly grown. © 2020 The Author(s).
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Protein corona fingerprinting to differentiate sepsis from non-infectious systemic inflammation
Papafilippou L., Claxton A., Dark P., Kostarelos K., Hadjidemetriou M. Nanoscale; 12 (18): 10240 - 10253. 2020. 10.1039/d0nr02788j. IF: 6.895
Rapid and accurate diagnosis of sepsis remains clinically challenging. The lack of specific biomarkers that can differentiate sepsis from non-infectious systemic inflammatory diseases often leads to excessive antibiotic treatment. Novel diagnostic tests are urgently needed to rapidly and accurately diagnose sepsis and enable effective treatment. Despite investment in cutting-edge technologies available today, the discovery of disease-specific biomarkers in blood remains extremely difficult. The highly dynamic environment of plasma restricts access to vital diagnostic information that can be obtained by proteomic analysis. Here, we employed clinically used lipid-based nanoparticles (AmBisome®) as an enrichment platform to analyze the human plasma proteome in the setting of sepsis. We exploited the spontaneous interaction of plasma proteins with nanoparticles (NPs) once in contact, called the 'protein corona', to discover previously unknown disease-specific biomarkers for sepsis diagnosis. Plasma samples obtained from non-infectious acute systemic inflammation controls and sepsis patients were incubated ex vivo with AmBisome® liposomes, and the resultant protein coronas were thoroughly characterised and compared by mass spectrometry (MS)-based proteomics. Our results demonstrate that the proposed nanoparticle enrichment technology enabled the discovery of 67 potential biomarker proteins that could reproducibly differentiate non-infectious acute systemic inflammation from sepsis. This study provides proof-of-concept evidence that nanoscale-based 'omics' enrichment technologies have the potential to substantially improve plasma proteomics analysis and to uncover novel biomarkers in a challenging clinical setting.
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Size-Dependent Pulmonary Impact of Thin Graphene Oxide Sheets in Mice: Toward Safe-by-Design
Rodrigues A.F., Newman L., Jasim D., Mukherjee S.P., Wang J., Vacchi I.A., Ménard-Moyon C., Bianco A., Fadeel B., Kostarelos K., Bussy C. Advanced Science; 7 (12, 1903200) 2020. 10.1002/advs.201903200. IF: 15.840
Safety assessment of graphene-based materials (GBMs) including graphene oxide (GO) is essential for their safe use across many sectors of society. In particular, the link between specific material properties and biological effects needs to be further elucidated. Here, the effects of lateral dimensions of GO sheets in acute and chronic pulmonary responses after single intranasal instillation in mice are compared. Micrometer-sized GO induces stronger pulmonary inflammation than nanometer-sized GO, despite reduced translocation to the lungs. Genome-wide RNA sequencing also reveals distinct size-dependent effects of GO, in agreement with the histopathological results. Although large GO, but not the smallest GO, triggers the formation of granulomas that persists for up to 90 days, no pulmonary fibrosis is observed. These latter results can be partly explained by Raman imaging, which evidences the progressive biotransformation of GO into less graphitic structures. The findings demonstrate that lateral dimensions play a fundamental role in the pulmonary response to GO, and suggest that airborne exposure to micrometer-sized GO should be avoided in the production plant or applications, where aerosolized dispersions are likely to occur. These results are important toward the implementation of a safer-by-design approach for GBM products and applications, for the benefit of workers and end-users. © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Splenic Capture and in Vivo Intracellular Biodegradation of Biological-Grade Graphene Oxide Sheets
Newman L., Jasim D.A., Prestat E., Lozano N., De Lazaro I., Nam Y., Assas B.M., Pennock J., Haigh S.J., Bussy C., Kostarelos K. ACS Nano; 14 (8): 10168 - 10186. 2020. 10.1021/acsnano.0c03438. IF: 14.588
Carbon nanomaterials, including 2D graphene-based materials, have shown promising applicability to drug delivery, tissue engineering, diagnostics, and various other biomedical areas. However, to exploit the benefits of these materials in some of the areas mentioned, it is necessary to understand their possible toxicological implications and long-term fate in vivo. We previously demonstrated that following intravenous administration, 2D graphene oxide (GO) nanosheets were largely excreted via the kidneys; however, a small but significant portion of the material was sequestered in the spleen. Herein, we interrogate the potential consequences of this accumulation and the fate of the spleen-residing GO over a period of nine months. We show that our thoroughly characterized GO materials are not associated with any detectable pathological consequences in the spleen. Using confocal Raman mapping of tissue sections, we determine the sub-organ biodistribution of GO at various time points after administration. The cells largely responsible for taking up the material are confirmed using immunohistochemistry coupled with Raman spectroscopy, and transmission electron microscopy (TEM). This combination of techniques identified cells of the splenic marginal zone as the main site of GO bioaccumulation. In addition, through analyses using both bright-field TEM coupled with electron diffraction and Raman spectroscopy, we reveal direct evidence of in vivo intracellular biodegradation of GO sheets with ultrastructural precision. This work offers critical information about biological processing and degradation of thin GO sheets by normal mammalian tissue, indicating that further development and exploitation of GO in biomedicine would be possible. Copyright © 2020 American Chemical Society.
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Stable, concentrated, biocompatible, and defect-free graphene dispersions with positive charge
Shin Y., Vranic S., Just-Baringo X., Gali S.M., Kisby T., Chen Y., Gkoutzidou A., Prestat E., Beljonne D., Larrosa I., Kostarelos K., Casiraghi C. Nanoscale; 12 (23): 12383 - 12394. 2020. 10.1039/d0nr02689a. IF: 6.895
The outstanding properties of graphene offer high potential for biomedical applications. In this framework, positively charged nanomaterials show better interactions with the biological environment, hence there is strong interest in the production of positively charged graphene nanosheets. Currently, production of cationic graphene is either time consuming or producing dispersions with poor stability, which strongly limit their use in the biomedical field. In this study, we made a family of new cationic pyrenes, and have used them to successfully produce water-based, highly concentrated, stable, and defect-free graphene dispersions with positive charge. The use of different pyrene derivatives as well as molecular dynamics simulations allowed us to get insights on the nanoscale interactions required to achieve efficient exfoliation and stabilisation. The cationic graphene dispersions show outstanding biocompatibility and cellular uptake as well as exceptional colloidal stability in the biological medium, making this material extremely attractive for biomedical applications. © 2020 The Royal Society of Chemistry.
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The biomolecule corona of lipid nanoparticles contains circulating cell-free DNA
Gardner L., Warrington J., Rogan J., Rothwell D.G., Brady G., Dive C., Kostarelos K., Hadjidemetriou M. Nanoscale Horizons; 5 (11): 1476 - 1486. 2020. 10.1039/d0nh00333f. IF: 9.927
The spontaneous adsorption of biomolecules onto the surface of nanoparticles (NPs) in complex physiological biofluids has been widely investigated over the last decade. Characterisation of the protein composition of the 'biomolecule corona' has dominated research efforts, whereas other classes of biomolecules, such as nucleic acids, have received no interest. Scarce, speculative statements exist in the literature about the presence of nucleic acids in the biomolecule corona, with no previous studies attempting to describe the contribution of genomic content to the blood-derived NP corona. Herein, we provide the first experimental evidence of the interaction of circulating cell-free DNA (cfDNA) with lipid-based NPs upon their incubation with human plasma samples, obtained from healthy volunteers and ovarian carcinoma patients. Our results also demonstrate an increased amount of detectable cfDNA in patients with cancer. Proteomic analysis of the same biomolecule coronas revealed the presence of histone proteins, suggesting an indirect, nucleosome-mediated NP-cfDNA interaction. The finding of cfDNA as part of the NP corona, offers a previously unreported new scope regarding the chemical composition of the 'biomolecule corona' and opens up new possibilities for the potential exploitation of the biomolecule corona for the enrichment and analysis of blood-circulating nucleic acids. © The Royal Society of Chemistry.
