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After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier\u2019s stability.<\/jats:p>","DOI":"10.1021\/acsabm.0c01209","type":"journal-article","created":{"date-parts":[[2020,12,23]],"date-time":"2020-12-23T10:52:48Z","timestamp":1608720768000},"page":"669-681","source":"Crossref","is-referenced-by-count":32,"title":["Real-Time Ratiometric Imaging of Micelles Assembly\nState in a Microfluidic Cancer-on-a-Chip"],"prefix":"10.1021","volume":"4","author":[{"given":"Natalia","family":"Feiner-Gracia","sequence":"first","affiliation":[{"name":"The Barcelona Institute of Science and Technology (BIST) , , Carrer Baldiri Reixac 15-21 , ,","place":["Barcelona, Spain, 08024"]},{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5612AZ"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adrianna","family":"Glinkowska Mares","sequence":"additional","affiliation":[{"name":"The Barcelona Institute of Science and Technology (BIST) , , Carrer Baldiri Reixac 15-21 , ,","place":["Barcelona, Spain, 08024"]},{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5612AZ"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Buzhor","sequence":"additional","affiliation":[{"name":"Tel-Aviv University , , ,","place":["Tel-Aviv, Israel, 6997801"]},{"name":"Tel-Aviv University , , ,","place":["Tel-Aviv, Israel, 6997801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Romen","family":"Rodriguez-Trujillo","sequence":"additional","affiliation":[{"name":"The Barcelona Institute of Science and Technology (BIST) , , Carrer Baldiri Reixac 15-21 , ,","place":["Barcelona, Spain, 08024"]},{"name":"University of Barcelona , , Carrer Marti\u0301 i Franque\u0300s 1 , ,","place":["Barcelona, Spain, 08028"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Josep","family":"Samitier Marti","sequence":"additional","affiliation":[{"name":"The Barcelona Institute of Science and Technology (BIST) , , Carrer Baldiri Reixac 15-21 , ,","place":["Barcelona, Spain, 08024"]},{"name":"University of Barcelona , , Carrer Marti\u0301 i Franque\u0300s 1 , ,","place":["Barcelona, Spain, 08028"]},{"name":"Biomaterials and Nanomedicine (CIBER-BBN) , , ,","place":["Madrid, Spain, 28029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8502-3302","authenticated-orcid":true,"given":"Roey J.","family":"Amir","sequence":"additional","affiliation":[{"name":"Tel-Aviv University , , ,","place":["Tel-Aviv, Israel, 6997801"]},{"name":"Tel-Aviv University , , ,","place":["Tel-Aviv, Israel, 6997801"]},{"name":"Tel-Aviv University , , ,","place":["Tel-Aviv, Israel, 6997801"]},{"name":"Tel-Aviv University , , ,","place":["Tel-Aviv, Israel, 6997801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia","family":"Pujals","sequence":"additional","affiliation":[{"name":"The Barcelona Institute of Science and Technology (BIST) , , Carrer Baldiri Reixac 15-21 , ,","place":["Barcelona, Spain, 08024"]},{"name":"University of Barcelona , , Carrer Marti\u0301 i Franque\u0300s 1 , ,","place":["Barcelona, Spain, 08028"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6837-0812","authenticated-orcid":true,"given":"Lorenzo","family":"Albertazzi","sequence":"additional","affiliation":[{"name":"The Barcelona Institute of Science and Technology (BIST) , , Carrer Baldiri Reixac 15-21 , ,","place":["Barcelona, Spain, 08024"]},{"name":"Eindhoven University of Technology , , ,","place":["Eindhoven, The Netherlands, 5612AZ"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,12,23]]},"reference":[{"issue":"14","key":"2026090222010906000_cit1","doi-asserted-by":"publisher","first-page":"1456","DOI":"10.1016\/j.addr.2006.09.011","article-title":"Nanomedicine: Developing Smarter Therapeutic and Diagnostic\nModalities","volume":"58","author":"Farokhzad","year":"2006","journal-title":"Adv. Drug Delivery Rev."},{"issue":"3","key":"2026090222010906000_cit2","doi-asserted-by":"publisher","first-page":"416","DOI":"10.1016\/j.ejps.2012.12.006","article-title":"A Review of Current Nanoparticle\nand Targeting Moieties for the Delivery of Cancer Therapeutics","volume":"48","author":"Steichen","year":"2013","journal-title":"Eur. J. Pharm. Sci."},{"issue":"5","key":"2026090222010906000_cit3","doi-asserted-by":"publisher","first-page":"294","DOI":"10.1016\/S0936-6555(05)80269-9","article-title":"Liposomal\nDoxorubicin (Doxil): An\nEffective New Treatment for Kaposi\u2019s Sarcoma in AIDS","volume":"6","author":"James","year":"1994","journal-title":"Clin. Oncol."},{"key":"2026090222010906000_cit4","doi-asserted-by":"publisher","first-page":"16014","DOI":"10.1038\/natrevmats.2016.14","article-title":"Analysis of Nanoparticle\nDelivery\nto Tumours","volume":"1","author":"Wilhelm","year":"2016","journal-title":"Nat. Rev. Mater."},{"issue":"11","key":"2026090222010906000_cit5","doi-asserted-by":"publisher","first-page":"653","DOI":"10.1038\/nrclinonc.2010.139","article-title":"Delivering Nanomedicine to Solid\nTumors","volume":"7","author":"Jain","year":"2010","journal-title":"Nat. Rev. Clin. Oncol."},{"issue":"1","key":"2026090222010906000_cit6","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1016\/j.nantod.2012.01.002","article-title":"Polymeric\nMicelle Stability","volume":"7","author":"Owen","year":"2012","journal-title":"Nano Today"},{"issue":"8","key":"2026090222010906000_cit7","doi-asserted-by":"publisher","first-page":"536","DOI":"10.1038\/nnano.2012.84","article-title":"Shear-Stress Sensitive\nLenticular\nVesicles for Targeted Drug Delivery","volume":"7","author":"Holme","year":"2012","journal-title":"Nat. Nanotechnol."},{"issue":"1","key":"2026090222010906000_cit8","doi-asserted-by":"publisher","first-page":"1600160","DOI":"10.1002\/mabi.201600160","article-title":"Physico-Chemical\nStrategies to Enhance Stability and Drug Retention of Polymeric Micelles\nfor Tumor-Targeted Drug Delivery","volume":"17","author":"Shi","year":"2017","journal-title":"Macromol.\nBiosci."},{"issue":"2","key":"2026090222010906000_cit9","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1016\/j.nantod.2014.04.008","article-title":"Challenges Associated with Penetration of Nanoparticles\nacross Cell and Tissue Barriers: A Review of Current Status and Future\nProspects","volume":"9","author":"Barua","year":"2014","journal-title":"Nano Today"},{"issue":"1","key":"2026090222010906000_cit10","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1016\/S0168-3659(99)00248-5","article-title":"Tumor Vascular Permeability\nand the EPR Effect in Macromolecular Therapeutics: A Review","volume":"65","author":"Maeda","year":"2000","journal-title":"J. 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Cancer"},{"issue":"10","key":"2026090222010906000_cit14","doi-asserted-by":"publisher","first-page":"5213","DOI":"10.1021\/la703570m","article-title":"Fast Release\nof Lipophilic Agents from Circulating\nPEG-PDLLA Micelles Revealed by in Vivo Fo\u0308rster Resonance Energy\nTransfer Imaging","volume":"24","author":"Chen","year":"2008","journal-title":"Langmuir"},{"issue":"15","key":"2026090222010906000_cit15","doi-asserted-by":"publisher","first-page":"6002","DOI":"10.1021\/ma200675w","article-title":"Stability\nof Self-Assembled Polymeric\nMicelles in Serum","volume":"44","author":"Lu","year":"2011","journal-title":"Macromolecules"},{"issue":"9","key":"2026090222010906000_cit16","doi-asserted-by":"publisher","first-page":"3133","DOI":"10.1021\/mp500329z","article-title":"FRET Imaging Approaches\nfor in Vitro and in Vivo Characterization of Synthetic Lipid Nanoparticles","volume":"11","author":"Gravier","year":"2014","journal-title":"Mol. Pharmaceutics"},{"issue":"37","key":"2026090222010906000_cit17","doi-asserted-by":"publisher","first-page":"7296","DOI":"10.1039\/C5SM01455G","article-title":"Nanoparticle\nStability in Biologically Relevant Media: Influence of Polymer Architecture","volume":"11","author":"Aguilar-Castillo","year":"2015","journal-title":"Soft Matter"},{"issue":"6","key":"2026090222010906000_cit18","doi-asserted-by":"publisher","first-page":"6179","DOI":"10.1021\/acsnano.8b02830","article-title":"The Blood Clearance\nKinetics and\nPathway of Polymeric Micelles in Cancer Drug Delivery","volume":"12","author":"Sun","year":"2018","journal-title":"ACS Nano"},{"issue":"46","key":"2026090222010906000_cit19","doi-asserted-by":"publisher","first-page":"16677","DOI":"10.1021\/jacs.7b08351","article-title":"Micellar Stability\nin Biological Media Dictates Internalization\nin Living Cells","volume":"139","author":"Feiner-Gracia","year":"2017","journal-title":"J. Am. Chem. Soc."},{"issue":"5","key":"2026090222010906000_cit20","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1016\/j.drudis.2012.10.003","article-title":"Three-Dimensional Cell Culture: The Missing Link in\nDrug Discovery","volume":"18","author":"Breslin","year":"2013","journal-title":"Drug Discovery Today"},{"issue":"1","key":"2026090222010906000_cit21","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1002\/btm2.10013","article-title":"Microfluidics-Based 3D Cell Culture Models: Utility in Novel Drug\nDiscovery and Delivery Research","volume":"1","author":"Gupta","year":"2016","journal-title":"Bioeng. Transl.\nMed."},{"issue":"2","key":"2026090222010906000_cit22","doi-asserted-by":"publisher","first-page":"e56910","DOI":"10.1371\/journal.pone.0056910","article-title":"In Vitro\nModel of Tumor Cell Extravasation","volume":"8","author":"Jeon","year":"2013","journal-title":"PLoS One"},{"key":"2026090222010906000_cit23","doi-asserted-by":"publisher","first-page":"34094","DOI":"10.1038\/srep34094","article-title":"Breast Cancer Cell\nInvasion into\na Three Dimensional Tumor-Stroma Microenvironment","volume":"6","author":"Truong","year":"2016","journal-title":"Sci. Rep."},{"issue":"10","key":"2026090222010906000_cit24","doi-asserted-by":"publisher","first-page":"e1004535","DOI":"10.1371\/journal.pcbi.1004535","article-title":"Cell Invasion\nDynamics into a Three Dimensional Extracellular Matrix Fibre Network","volume":"11","author":"Kim","year":"2015","journal-title":"PLoS Comput. Biol."},{"issue":"6","key":"2026090222010906000_cit25","doi-asserted-by":"publisher","first-page":"969","DOI":"10.1007\/s10404-012-1104-6","article-title":"A Three-Dimensional\nMicrofluidic Tumor Cell Migration Assay to Screen the Effect of Anti-Migratory\nDrugs and Interstitial Flow","volume":"14","author":"Kalchman","year":"2013","journal-title":"Microfluid. Nanofluid."},{"issue":"22","key":"2026090222010906000_cit26","doi-asserted-by":"publisher","first-page":"8454","DOI":"10.1021\/ac202170e","article-title":"Sprouting Angiogenesis under a Chemical Gradient Regulated by Interactions\nwith an Endothelial Monolayer in a Microfluidic Platform","volume":"83","author":"Jeong","year":"2011","journal-title":"Anal. Chem."},{"issue":"5","key":"2026090222010906000_cit27","doi-asserted-by":"publisher","first-page":"e37333","DOI":"10.1371\/journal.pone.0037333","article-title":"Ensemble Analysis\nof Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures","volume":"7","author":"Farahat","year":"2012","journal-title":"PLoS One"},{"issue":"17","key":"2026090222010906000_cit28","doi-asserted-by":"publisher","first-page":"2822","DOI":"10.1039\/C9LC00140A","article-title":"Tumor Spheroid-on-a-Chip: A Standardized\nMicrofluidic\nCulture Platform for Investigating Tumor Angiogenesis","volume":"19","author":"Ko","year":"2019","journal-title":"Lab Chip"},{"key":"2026090222010906000_cit29","doi-asserted-by":"publisher","first-page":"119547","DOI":"10.1016\/j.biomaterials.2019.119547","article-title":"Vascularized\nCancer on a Chip: The\nEffect of Perfusion on Growth and Drug Delivery of Tumor Spheroid","volume":"229","author":"Nashimoto","year":"2020","journal-title":"Biomaterials"},{"issue":"1","key":"2026090222010906000_cit30","doi-asserted-by":"publisher","first-page":"707","DOI":"10.1038\/s41598-017-00750-3","article-title":"A Facile Method to Probe the Vascular Permeability\nof Nanoparticles in Nanomedicine Applications","volume":"7","author":"Ho","year":"2017","journal-title":"Sci. Rep."},{"issue":"1","key":"2026090222010906000_cit31","doi-asserted-by":"publisher","first-page":"9359","DOI":"10.1038\/s41598-017-09815-9","article-title":"A Biomimetic Microfluidic Tumor Microenvironment Platform\nMimicking the EPR Effect for Rapid Screening of Drug Delivery Systems","volume":"7","author":"Tang","year":"2017","journal-title":"Sci. Rep."},{"issue":"3","key":"2026090222010906000_cit32","doi-asserted-by":"publisher","first-page":"808","DOI":"10.1021\/ac901560w","article-title":"Technique\nfor Real-Time Measurements of Endothelial Permeability in a Microfluidic\nMembrane Chip Using Laser-Induced Fluorescence Detection","volume":"82","author":"Young","year":"2010","journal-title":"Anal. Chem."},{"issue":"3","key":"2026090222010906000_cit33","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1038\/nrm1858","article-title":"Capturing Complex 3D Tissue Physiology in Vitro","volume":"7","author":"Griffith","year":"2006","journal-title":"Nat. Rev. Mol. Cell Biol."},{"issue":"7","key":"2026090222010906000_cit34","doi-asserted-by":"publisher","first-page":"1256","DOI":"10.1016\/j.biotechadv.2014.07.009","article-title":"Three-Dimensional in Vitro Tumor\nModels for Cancer Research and Drug Evaluation","volume":"32","author":"Xu","year":"2014","journal-title":"Biotechnol. Adv."},{"issue":"6","key":"2026090222010906000_cit35","doi-asserted-by":"publisher","first-page":"1490","DOI":"10.1002\/bit.21698","article-title":"A Perfusable 3D\nCell\u2013Matrix Tissue Culture Chamber\nfor in Situ Evaluation of Nanoparticle Vehicle Penetration and Transport","volume":"99","author":"Ng","year":"2008","journal-title":"Biotechnol. Bioeng."},{"key":"2026090222010906000_cit36","doi-asserted-by":"publisher","first-page":"2718","DOI":"10.1038\/ncomms3718","article-title":"Tumour-on-a-Chip Provides an Optical\nWindow into Nanoparticle\nTissue Transport","volume":"4","author":"Albanese","year":"2013","journal-title":"Nat. Commun."},{"issue":"34","key":"2026090222010906000_cit37","doi-asserted-by":"publisher","first-page":"4947","DOI":"10.1039\/C7PY00559H","article-title":"Multicellular\nTumor Spheroids: A\nRelevant 3D Model for the in Vitro Preclinical Investigation of Polymer\nNanomedicines","volume":"8","author":"Lazzari","year":"2017","journal-title":"Polym. Chem."},{"issue":"5","key":"2026090222010906000_cit38","doi-asserted-by":"publisher","first-page":"566","DOI":"10.1038\/s41563-019-0566-2","article-title":"The Entry of\nNanoparticles into Solid Tumours","volume":"19","author":"Sindhwani","year":"2020","journal-title":"Nat. Mater."},{"issue":"11","key":"2026090222010906000_cit39","doi-asserted-by":"publisher","first-page":"11600","DOI":"10.1021\/acsnano.8b06846","article-title":"Tumor-Vasculature-on-a-Chip for Investigating\nNanoparticle Extravasation and Tumor Accumulation","volume":"12","author":"Wang","year":"2018","journal-title":"ACS Nano"},{"key":"2026090222010906000_cit40","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1016\/j.aca.2018.06.038","article-title":"A Novel 3D Breast-Cancer-on-Chip\nPlatform for Therapeutic Evaluation of Drug Delivery Systems","volume":"1036","author":"Chen","year":"2018","journal-title":"Anal. Chim. Acta"},{"key":"2026090222010906000_cit41","doi-asserted-by":"publisher","first-page":"157","DOI":"10.1016\/j.jconrel.2014.08.027","article-title":"Simulation\nof Complex Transport of Nanoparticles around a Tumor Using Tumor-Microenvironment-on-Chip","volume":"194","author":"Kwak","year":"2014","journal-title":"J. Controlled Release"},{"issue":"1","key":"2026090222010906000_cit42","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1002\/jcp.24683","article-title":"3D Cell\nCulture Systems: Advantages and Applications","volume":"230","author":"Ravi","year":"2015","journal-title":"J. Cell. Physiol."},{"issue":"2","key":"2026090222010906000_cit43","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1016\/j.jconrel.2010.04.007","article-title":"Flow and Adhesion of Drug Carriers in Blood Vessels\nDepend on Their Shape: A Study Using Model Synthetic Microvascular\nNetworks","volume":"146","author":"Doshi","year":"2010","journal-title":"J. Controlled Release"},{"issue":"10","key":"2026090222010906000_cit44","doi-asserted-by":"publisher","first-page":"839","DOI":"10.1038\/nrm2236","article-title":"The Third Dimension Bridges the Gap\nbetween Cell Culture and Live Tissue","volume":"8","author":"Pampaloni","year":"2007","journal-title":"Nat. Rev.\nMol. 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