title: Cryo-soft X-ray tomography as a quantitative three-dimensional tool to model nanoparticle:cell interaction.
abstract: BACKGROUND
  Recent advances in nanoparticle design have generated new possibilities for nano-biotechnology and nano-medicine. Here we used cryo-soft X-ray tomography (cryo-SXT) to collect comprehensive three-dimensional (3D) data to characterise the interaction of superparamagnetic iron oxide nanoparticles (SPION) with a breast cancer cell line.
  
  
  RESULTS
  We incubated MCF-7 (a human breast cancer cell line) from 0 to 24 h with SPION (15 nm average diameter, coated with dimercaptosuccinic acid), a system that has been studied previously using various microscopy and bulk techniques. This system facilitates the validation and contextualization of the new 3D data acquired using the cryo-SXT-based approach. After vitrification, samples tested by correlative cryo-epifluorescent microscopy showed SPION accumulation in acidic vesicles related to the endocytic pathway. Microscopy grids bearing MCF-7 cells were then analysed by cryo-SXT to generate whole cell volume 3D maps. Cryo-SXT is an emerging technique that benefits from high X-ray penetration into the biological material to image close-to-native vitrified cells at nanometric resolution with no chemical fixation or staining agents. This unique possibility of obtaining 3D information from whole cells allows quantitative statistical analysis of SPION-containing vesicle (SCV) accumulation inside cells, including vesicle number and size, distances between vesicles, and their distance from the nucleus.
  
  
  CONCLUSIONS
  Correlation between fluorescent microscopy, cryo-SXT and transmission electron microscopy allowed us to identify SCV and to generate 3D data for statistical analysis of SPION:cell interaction. This study supports continuous transfer of the internalized SPION from the plasma membrane to an accumulation area near the cell nucleus. Statistical analysis showed SCV increase in number and size concomitant with longer incubation times, and therefore an increase in their accumulated volume within the cell. This cumulative effect expands the accumulation area and cell organelles such as mitochondria are consequently displaced to the periphery. Our 3D cryo-SXT approach demonstrates that a comprehensive quantitative description of SPION:cell interaction is possible, which will serve as a basis for metal-based nanoparticle design and for selection of those best suited for hyperthermia treatment, drug delivery and image diagnosis in nanobiomedicine.
authors.affiliation: Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049, Spain, Madrid. m.chiappi@imperial.ac.uk.|National Heart and Lung Institute, Imperial College London, Exhibition Road, London, SW7 2AZ, UK. m.chiappi@imperial.ac.uk.
authors.forename: Michele
authors.initials: M
authors.lastname: Chiappi
authors.affiliation: Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049, Spain, Madrid. jj.conesa@cnb.csic.es.
authors.forename: José Javier
authors.initials: JJ
authors.lastname: Conesa
authors.affiliation: MISTRAL Beamline-Experiments Division, ALBA Synchrotron Light Source, Cerdanyola del Vallès, 08290, Barcelona, Spain. epereiro@cells.es.
authors.forename: Eva
authors.initials: E
authors.lastname: Pereiro
authors.affiliation: Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049, Spain, Madrid. coss@cnb.csic.es.
authors.forename: Carlos Oscar Sánchez
authors.initials: CO
authors.lastname: Sorzano
authors.affiliation: Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049, Spain, Madrid. mjrguez@cnb.csic.es.
authors.forename: María Josefa
authors.initials: MJ
authors.lastname: Rodríguez
authors.affiliation: Laboratory for Synchrotron Radiation-Catalysis and Sustainable Chemistry, Paul Scherrer Institut (PSI), 5232, Villigen, Switzerland. katja.henzler@psi.ch.
authors.forename: Katja
authors.initials: K
authors.lastname: Henzler
authors.affiliation: Institute for Soft Matter and Functional Materials, Microscopy Group Electron Storage Ring BESSY II, Helmholtz-Zentrum Berlin, Albert-Einstein-Str. 15, 12489, Berlin, Germany. gerd.schneider@helmholtz-berlin.de.
authors.forename: Gerd
authors.initials: G
authors.lastname: Schneider
authors.affiliation: Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049, Spain, Madrid. fjchichon@cnb.csic.es.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Chichón
authors.affiliation: Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049, Spain, Madrid. jlcarras@cnb.csic.es.|Unidad Asociada CNB-Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), Cantoblanco, 28049, Madrid, Spain. jlcarras@cnb.csic.es.
authors.forename: José L
authors.initials: JL
authors.lastname: Carrascosa
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002477:Cells / Q000378:metabolism*; D015925:Cryopreservation / Q000379:methods; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence / Q000379:methods; D053758:Nanoparticles / Q000378:metabolism*; D014056:Tomography, X-Ray / Q000379:methods