{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T14:51:18Z","timestamp":1791471078439,"version":"4.3.4"},"reference-count":46,"publisher":"American Chemical Society (ACS)","issue":"40","license":[{"start":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:00:00Z","timestamp":1790294400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"Narodowe Centrum Nauki","doi-asserted-by":"publisher","award":["2021\/41\/B\/ST4\/00464"],"award-info":[{"award-number":["2021\/41\/B\/ST4\/00464"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Understanding whether internalized nanoparticles remain confined within intracellular vesicles or are released into the cytosol is essential for evaluating their biological activity and potential use as delivery systems. However, this distinction is often difficult to establish by using fluorescence imaging alone, particularly at nanomolar probe concentrations. Here, we propose fluorescence correlation spectroscopy (FCS) combined with length-scale-dependent viscosity analysis and appropriate diffusion models as an approach for assessing nanoparticle uptake and intracellular localization. The method was evaluated in HeLa cells using fluorescent polystyrene nanoparticles, cell-penetrating peptide-coated nanoparticles, dextran of two molecular weights, and receptor-internalized transferrin. Cytosolic probes were identified by autocorrelation curves that fit an anomalous diffusion model and by diffusion coefficients consistent with those predicted by the cytoplasmic length scale-dependent viscosity profile. In contrast, vesicle-associated probes produced characteristic two-step autocorrelation curves that were modeled as the sum of probe diffusion within a confined space, vesicle translation, and directed intracellular transport. Bare nanoparticles, pinocytosed dextrans, and transferrin displayed predominantly vesicle-associated mobility, whereas microinjected dextrans and cell-penetrating-peptide-associated nanoparticles exhibited diffusion in the cytosol. Our results demonstrate that FCS can complement fluorescence imaging by detecting cytosolic probe populations and distinguishing between cytosolic release and vesicular retention.<\/jats:p>","DOI":"10.1021\/acs.jpcb.6c05465","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T12:02:04Z","timestamp":1790337724000},"page":"10477-10487","source":"Crossref","is-referenced-by-count":0,"title":["Distinguishing\nFree Cytosolic and Vesicle-Confined\nNanoparticle Motion by Fluorescence Correlation Spectroscopy"],"prefix":"10.1021","volume":"130","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7396-9571","authenticated-orcid":true,"given":"Karina","family":"Kwapiszewska","sequence":"first","affiliation":[{"name":"Polish Academy of Sciences , , Kasprzaka 44\/52 , ,","place":["Warsaw, Poland, 01-224"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Grzegorz","family":"Bubak","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , Kasprzaka 44\/52 , ,","place":["Warsaw, Poland, 01-224"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6023-5499","authenticated-orcid":true,"given":"Krzysztof","family":"Bielec","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , Kasprzaka 44\/52 , ,","place":["Warsaw, Poland, 01-224"]},{"name":"University of Zurich , , Winterthurerstrasse 190 , ,","place":["Zurich, Switzerland, CH-8057"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3307-2882","authenticated-orcid":true,"given":"Aneta","family":"Magiera","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , Kasprzaka 44\/52 , ,","place":["Warsaw, Poland, 01-224"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alicja","family":"Zgorzelska","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , Kasprzaka 44\/52 , ,","place":["Warsaw, Poland, 01-224"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert","family":"Ho\u0142yst","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , Kasprzaka 44\/52 , ,","place":["Warsaw, Poland, 01-224"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,25]]},"reference":[{"key":"2026100809091114000_cit1","doi-asserted-by":"publisher","first-page":"679","DOI":"10.2174\/1389201022666210714145356","article-title":"Cellular Uptake Pathways of Nanoparticles: Process\nof Endocytosis and Factors Affecting Their Fate","volume":"23","author":"Varma","year":"2022","journal-title":"Curr. Pharm. Biotechnol."},{"key":"2026100809091114000_cit2","doi-asserted-by":"publisher","first-page":"1059","DOI":"10.1186\/s12889-023-15958-4","article-title":"Human and Environmental\nImpacts of Nanoparticles: A Scoping Review\nof the Current Literature","volume":"23","author":"Kumah","year":"2023","journal-title":"BMC Public Health"},{"key":"2026100809091114000_cit3","doi-asserted-by":"publisher","first-page":"2674","DOI":"10.1039\/D2NA00534D","article-title":"The Toxicity of Nanoparticles and\nTheir Interaction\nwith Cells: An in Vitro Metabolomic Perspective","volume":"5","author":"Awashra","year":"2023","journal-title":"Nanoscale Adv."},{"key":"2026100809091114000_cit4","doi-asserted-by":"publisher","first-page":"157","DOI":"10.1186\/s11671-023-03943-0","article-title":"Exploration\nof Inorganic Nanoparticles for Revolutionary Drug Delivery Applications:\nA Critical Review","volume":"18","author":"Unnikrishnan","year":"2023","journal-title":"Discover Nano"},{"key":"2026100809091114000_cit5","doi-asserted-by":"publisher","first-page":"587","DOI":"10.1186\/s12951-024-02859-w","article-title":"Redox-Manipulating Nanocarriers for\nAnticancer Drug Delivery: A Systematic\nReview","volume":"22","author":"Meng","year":"2024","journal-title":"J. Nanobiotechnol."},{"key":"2026100809091114000_cit6","doi-asserted-by":"publisher","first-page":"120618","DOI":"10.1016\/j.jenvman.2024.120618","article-title":"Microplastics\nand Nanoplastics: Source, Behavior, Remediation, and Multi-Level Environmental\nImpact","volume":"356","author":"Arif","year":"2024","journal-title":"J. Environ. Manage."},{"key":"2026100809091114000_cit7","doi-asserted-by":"publisher","first-page":"1512","DOI":"10.3390\/nano13091512","article-title":"Risk Assessment of Nanomaterials Toxicity","volume":"13","author":"Hartwig","year":"2023","journal-title":"Nanomaterials"},{"key":"2026100809091114000_cit8","doi-asserted-by":"publisher","first-page":"1926","DOI":"10.3390\/ijms25031926","article-title":"Zebrafish\nInsights into Nanomaterial Toxicity: A Focused Exploration on Metallic,\nMetal Oxide, Semiconductor, and Mixed-Metal Nanoparticles","volume":"25","author":"Mutalik","year":"2024","journal-title":"Int. J. Mol. Sci."},{"key":"2026100809091114000_cit9","doi-asserted-by":"publisher","first-page":"2215","DOI":"10.3390\/nano13152215","article-title":"Exocytosis\nof Nanoparticles: A Comprehensive Review","volume":"13","author":"Liu","year":"2023","journal-title":"Nanomaterials"},{"key":"2026100809091114000_cit10","doi-asserted-by":"publisher","first-page":"4218","DOI":"10.1039\/C6CS00636A","article-title":"Cellular Uptake of Nanoparticles: Journey inside the\nCell","volume":"46","author":"Behzadi","year":"2017","journal-title":"Chem. Soc. 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Drug Delivery Rev."},{"key":"2026100809091114000_cit13","doi-asserted-by":"publisher","first-page":"15743","DOI":"10.1039\/D0NR01627F","article-title":"Quantifying the\nLevel of Nanoparticle Uptake in Mammalian Cells Using Flow Cytometry","volume":"12","author":"Shin","year":"2020","journal-title":"Nanoscale"},{"key":"2026100809091114000_cit14","doi-asserted-by":"publisher","first-page":"2196","DOI":"10.1039\/D0NA00716A","article-title":"Kinetics of\nNanoparticle Uptake into and Distribution in Human Cells","volume":"3","author":"\u00c5berg","year":"2021","journal-title":"Nanoscale Adv."},{"key":"2026100809091114000_cit15","doi-asserted-by":"publisher","first-page":"14730","DOI":"10.1039\/C7NR04640E","article-title":"Influence of Surface Coating on the\nIntracellular Behaviour of Gold Nanoparticles: A Fluorescence Correlation\nSpectroscopy Study","volume":"9","author":"Silvestri","year":"2017","journal-title":"Nanoscale"},{"key":"2026100809091114000_cit16","doi-asserted-by":"publisher","first-page":"140454","DOI":"10.1098\/rsos.140454","article-title":"Photothermal Raster Image Correlation\nSpectroscopy of Gold Nanoparticles in Solution and on Live Cells","volume":"2","author":"Nieves","year":"2015","journal-title":"R. Soc. Open Sci."},{"key":"2026100809091114000_cit17","doi-asserted-by":"publisher","first-page":"1424","DOI":"10.3390\/ijms20061424","article-title":"Two-Dimensional\nand Three-Dimensional Single Particle Tracking of Upconverting Nanoparticles\nin Living Cells","volume":"20","author":"Shin","year":"2019","journal-title":"Int. J. Mol. Sci."},{"key":"2026100809091114000_cit18","doi-asserted-by":"publisher","first-page":"10436","DOI":"10.1039\/D1NR00901J","article-title":"Imaging of Nanoparticle Uptake and\nKinetics of Intracellular Trafficking in Individual Cells","volume":"13","author":"Vtyurina","year":"2021","journal-title":"Nanoscale"},{"key":"2026100809091114000_cit19","doi-asserted-by":"publisher","first-page":"1830","DOI":"10.1038\/s41467-018-04185-w","article-title":"Single Quantum Dot Tracking Reveals\nthe Impact of Nanoparticle Surface on Intracellular State","volume":"9","author":"Zahid","year":"2018","journal-title":"Nat. Commun."},{"key":"2026100809091114000_cit20","doi-asserted-by":"publisher","first-page":"9831","DOI":"10.1021\/acs.jpcb.7b07158","article-title":"Apparent Anomalous Diffusion in the\nCytoplasm of Human Cells: The Effect of Probes\u2019 Polydispersity","volume":"121","author":"Kalwarczyk","year":"2017","journal-title":"J. Phys. Chem. B"},{"key":"2026100809091114000_cit21","doi-asserted-by":"publisher","first-page":"10","DOI":"10.1039\/D0NA00454E","article-title":"Nanoscopy\nfor Endosomal Escape Quantification","volume":"3","author":"Andrian","year":"2021","journal-title":"Nanoscale\nAdv."},{"key":"2026100809091114000_cit22","doi-asserted-by":"publisher","first-page":"1379","DOI":"10.1021\/acscentsci.8b00446","article-title":"Fluorescence\nCorrelation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein\nCargo by Cell-Permeant Miniature Proteins","volume":"4","author":"Wissner","year":"2018","journal-title":"ACS\nCent. 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Lett."},{"key":"2026100809091114000_cit26","doi-asserted-by":"publisher","first-page":"5906","DOI":"10.1038\/s41598-019-42418-0","article-title":"Determination\nof Oligomerization State of Drp1 Protein in Living Cells at Nanomolar\nConcentrations","volume":"9","author":"Kwapiszewska","year":"2019","journal-title":"Sci. Rep."},{"key":"2026100809091114000_cit27","doi-asserted-by":"publisher","first-page":"266","DOI":"10.1038\/s41565-021-00858-8","article-title":"Key Principles and Methods for Studying\nthe Endocytosis of Biological and Nanoparticle Therapeutics","volume":"16","author":"Rennick","year":"2021","journal-title":"Nat. Nanotechnol."},{"key":"2026100809091114000_cit28","doi-asserted-by":"publisher","first-page":"662","DOI":"10.1021\/nl052396o","article-title":"Determining\nthe Size and Shape Dependence\nof Gold Nanoparticle Uptake into Mammalian Cells","volume":"6","author":"Chithrani","year":"2006","journal-title":"Nano Lett."},{"key":"2026100809091114000_cit29","doi-asserted-by":"publisher","first-page":"8655","DOI":"10.1021\/acsnano.5b03184","article-title":"Physical Principles\nof Nanoparticle Cellular Endocytosis","volume":"9","author":"Zhang","year":"2015","journal-title":"ACS Nano"},{"key":"2026100809091114000_cit30","doi-asserted-by":"publisher","first-page":"570","DOI":"10.1186\/s12964-024-01929-8","article-title":"A Novel\nProcess for Transcellular Hemoglobin Transport from Macrophages to\nCancer Cells","volume":"22","author":"Braniewska","year":"2024","journal-title":"Cell Commun. Signaling"},{"key":"2026100809091114000_cit31","doi-asserted-by":"publisher","first-page":"7131","DOI":"10.1039\/D1AN01769A","article-title":"Quantitative Analysis of Biochemical Processes in Living\nCells at a Single-Molecule Level: A Case of Olaparib\u2013PARP1\n(DNA Repair Protein) Interactions","volume":"146","author":"Karpin\u0301ska","year":"2021","journal-title":"Analyst"},{"key":"2026100809091114000_cit32","doi-asserted-by":"publisher","first-page":"16486","DOI":"10.1038\/s41598-019-52758-6","article-title":"Stability of Cytoplasmic Nanoviscosity\nduring Cell Cycle of HeLa Cells Synchronized with Aphidicolin","volume":"9","author":"Szczepan\u0301ski","year":"2019","journal-title":"Sci. Rep."},{"key":"2026100809091114000_cit33","doi-asserted-by":"publisher","first-page":"1127","DOI":"10.1038\/ncomms2114","article-title":"Measuring\nthe Size of Individual\nParticles from Three-Dimensional Imaging Experiments","volume":"3","author":"Kurita","year":"2012","journal-title":"Nat. Commun."},{"key":"2026100809091114000_cit34","doi-asserted-by":"publisher","first-page":"7366","DOI":"10.1039\/c1sm05217a","article-title":"Scale-Dependent\nDiffusion of Spheres in Solutions of\nFlexible and Rigid Polymers: Mean Square Displacement and Autocorrelation\nFunction for FCS and DLS Measurements","volume":"7","author":"Ochab-Marcinek","year":"2011","journal-title":"Soft\nMatter"},{"key":"2026100809091114000_cit35","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1016\/j.ceb.2007.11.006","article-title":"Cargo Transport:\nMolecular Motors\nNavigate a Complex Cytoskeleton","volume":"20","author":"Ross","year":"2008","journal-title":"Curr. Opin.\nCell Biol."},{"key":"2026100809091114000_cit36","doi-asserted-by":"publisher","first-page":"arXiv:2603.29684","DOI":"10.48550\/arXiv.2603.29684","article-title":"FcsIT: An\nOpen-Source, Cross-Platform Tool for Correlation and Analysis of Fluorescence\nCorrelation Spectroscopy Data","author":"Kalwarczyk","year":"2026","journal-title":"arXiv"},{"key":"2026100809091114000_cit37","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1016\/j.jcis.2022.07.040","article-title":"Entanglement\nof Polymer Chains in Hypertonic Medium Enhances the Delivery of DNA\nand Other Biomacromolecules into Cells","volume":"627","author":"Karpin\u0301ska","year":"2022","journal-title":"J. Colloid\nInterface Sci."},{"key":"2026100809091114000_cit38","doi-asserted-by":"publisher","first-page":"2258","DOI":"10.1073\/pnas.80.8.2258","article-title":"pH and the Recycling of Transferrin\nduring Receptor-Mediated Endocytosis","volume":"80","author":"Dautry-Varsat","year":"1983","journal-title":"Proc.\nNatl. Acad. Sci. U.S.A."},{"key":"2026100809091114000_cit39","doi-asserted-by":"publisher","first-page":"638","DOI":"10.1038\/nbt.2612","article-title":"Image-Based Analysis\nof Lipid Nanoparticle-Mediated siRNA Delivery, Intracellular Trafficking\nand Endosomal Escape","volume":"31","author":"Gilleron","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"2026100809091114000_cit40","doi-asserted-by":"publisher","first-page":"3294","DOI":"10.1016\/S0006-3495(00)76561-1","article-title":"Fluorescence Correlation Spectroscopy in Small Cytosolic\nCompartments Depends Critically on the Diffusion Model Used","volume":"79","author":"Gennerich","year":"2000","journal-title":"Biophys. J."},{"key":"2026100809091114000_cit41","doi-asserted-by":"publisher","first-page":"1260","DOI":"10.1529\/biophysj.103.036483","article-title":"Spatial-Temporal\nStudies of Membrane Dynamics: Scanning Fluorescence Correlation Spectroscopy","volume":"87","author":"Ruan","year":"2004","journal-title":"Biophys. J."},{"key":"2026100809091114000_cit42","doi-asserted-by":"publisher","first-page":"1317","DOI":"10.1529\/biophysj.105.062836","article-title":"Measuring Fast Dynamics\nin Solutions and Cells with\na Laser Scanning Microscope","volume":"89","author":"Digman","year":"2005","journal-title":"Biophys. J."},{"key":"2026100809091114000_cit43","doi-asserted-by":"publisher","first-page":"169","DOI":"10.1111\/jmi.12080","article-title":"A Comparison\nof Single Particle Tracking and Temporal\nImage Correlation Spectroscopy for Quantitative Analysis of Endosome\nMotility","volume":"252","author":"Lund","year":"2013","journal-title":"J. Microsc."},{"key":"2026100809091114000_cit44","doi-asserted-by":"publisher","first-page":"14615","DOI":"10.1039\/D3NR02838K","article-title":"A Super-Resolution and Transmission Electron Microscopy\nCorrelative Approach to Study Intracellular Trafficking of Nanoparticles","volume":"15","author":"Andrian","year":"2023","journal-title":"Nanoscale"},{"key":"2026100809091114000_cit45","doi-asserted-by":"publisher","first-page":"102872","DOI":"10.1016\/j.nano.2025.102872","article-title":"3D Correlative\nLight and Electron Microscopy Reveals the Uptake and Processing of\nInorganic-Organic Hybrid Nanoparticles into Cancer Cells","volume":"70","author":"Herbsleb","year":"2025","journal-title":"Nanomedicine"},{"key":"2026100809091114000_cit46","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2307800120","article-title":"Endosomal Escape: A Bottleneck for\nLNP-Mediated Therapeutics","volume":"121","author":"Chatterjee","year":"2024","journal-title":"Proc. Natl. Acad.\nSci. U.S.A."}],"container-title":["The Journal of Physical\nChemistry B"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jpcb.6c05465","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/130\/40\/10477\/68649476\/acs.jpcb.6c05465.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/130\/40\/10477\/68649476\/acs.jpcb.6c05465.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T14:03:07Z","timestamp":1791468187000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article\/130\/40\/10477\/5438313\/Distinguishing-Free-Cytosolic-and-Vesicle-Confined"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,25]]},"references-count":46,"journal-issue":{"issue":"40","published-online":{"date-parts":[[2026,9,25]]},"published-print":{"date-parts":[[2026,10,8]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jpcb.6c05465","relation":{},"ISSN":["1520-6106","1520-5207"],"issn-type":[{"value":"1520-6106","type":"print"},{"value":"1520-5207","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,25]]}}}