{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T21:54:02Z","timestamp":1790114042538,"version":"4.0.1"},"reference-count":36,"publisher":"Public Library of Science (PLoS)","issue":"3","license":[{"start":{"date-parts":[[2025,3,25]],"date-time":"2025-03-25T00:00:00Z","timestamp":1742860800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100021772","name":"Deanship of Scientific Research, The University of Jordan","doi-asserted-by":"crossref","award":["2446"],"award-info":[{"award-number":["2446"]}],"id":[{"id":"10.13039\/501100021772","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The distinctive physiological and physical properties of 3D cultures that mimic tumor microenvironments <jats:italic>in vivo<\/jats:italic> make them more suitable for assessing the efficacy of drugs and nanoparticles compared to 2D culture models. Therefore, this study aims to examine and contrast how liposomes interact with cell cultures in both 2D and 3D models. Hanging drop technique was used to generate 3D spheroids. Cellular toxicity of Doxorubicin and Doxil<jats:sup>\u00ae<\/jats:sup>-liposomes was tested using an MTT assay. Cellular uptake of Doxil<jats:sup>\u00ae<\/jats:sup>-liposomes was investigated in 3D and 2D cell culture models using flow cytometry and confocal microscopy. Finally, migration and invasion assays were used to investigate the Doxil<jats:sup>\u00ae<\/jats:sup>-liposomes interaction with the two models 2D model and 3D model, respectively. Our findings show that cells were able to form spheroid structures when a specific cell ratio was maintained. Flow cytometry analysis revealed that 2D cells exhibited higher Doxil<jats:sup>\u00ae<\/jats:sup>-liposome uptake than 3D cells. The data obtained from confocal and fluorescent microscopy supported the findings of the flow cytometry analysis. Furthermore, the MTT assay showed that Doxil<jats:sup>\u00ae<\/jats:sup>-liposomes induced less metabolic-disruption compared to free Doxorubicin. Our results also demonstrated that Doxil<jats:sup>\u00ae<\/jats:sup>-liposomes interacted more loosely with the 3D model than 2D cells, which was further confirmed by measurements of the total migration and invasion areas. Therefore, a 3D model replicating the <jats:italic>in vivo<\/jats:italic> conditions of tumor structure and extracellular matrix to assess the delivery of liposomal-nanoparticles to spheroids through a collagen matrix can be more informative and recapitulate the <jats:italic>in vivo<\/jats:italic> microenvironment than the 2D model.<\/jats:p>","DOI":"10.1371\/journal.pone.0320374","type":"journal-article","created":{"date-parts":[[2025,3,25]],"date-time":"2025-03-25T19:26:06Z","timestamp":1742930766000},"page":"e0320374","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":7,"title":["In vitro cellular interaction of drug-loaded liposomes with 2D and 3D cell culture of U87-MG cell line"],"prefix":"10.1371","volume":"20","author":[{"given":"Tasneem","family":"Alsheleh","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manar","family":"Zraikat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fadwa","family":"Daoud","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dana A.","family":"Alqudah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7446-4436","authenticated-orcid":true,"given":"Sharif","family":"Abdelghany","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1175-5017","authenticated-orcid":true,"given":"Ahmed","family":"Abu Siniyeh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2946-7328","authenticated-orcid":true,"given":"Walhan","family":"Alshaer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,3,25]]},"reference":[{"issue":"7","key":"pone.0320374.ref001","doi-asserted-by":"crossref","first-page":"1256","DOI":"10.1016\/j.biotechadv.2014.07.009","article-title":"Three-dimensional in vitro tumor models for cancer research and drug evaluation","volume":"32","author":"X Xu","year":"2014","journal-title":"Biotechnol Adv"},{"issue":"2","key":"pone.0320374.ref002","doi-asserted-by":"crossref","first-page":"133","DOI":"10.2147\/IJN.S596","article-title":"Drug delivery and nanoparticles:applications and hazards","volume":"3","author":"WH De Jong","year":"2008","journal-title":"Int J Nanomedicine"},{"issue":"7","key":"pone.0320374.ref003","doi-asserted-by":"crossref","first-page":"1841","DOI":"10.1016\/j.nano.2015.06.007","article-title":"In vitro experiments showing enhanced release of doxorubicin from Doxil\u00ae in the presence of ammonia may explain drug release at tumor site","volume":"11","author":"L Silverman","year":"2015","journal-title":"Nanomedicine"},{"issue":"22","key":"pone.0320374.ref004","doi-asserted-by":"crossref","first-page":"12200","DOI":"10.3390\/ijms222212200","article-title":"3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages","volume":"22","author":"O Habanjar","year":"2021","journal-title":"Int J Mol Sci"},{"key":"pone.0320374.ref005","doi-asserted-by":"crossref","first-page":"891673","DOI":"10.3389\/fonc.2022.891673","article-title":"Implications of Three-Dimensional Cell Culture in Cancer Therapeutic Research","volume":"12","author":"K Poornima","year":"2022","journal-title":"Front Oncol"},{"issue":"3","key":"pone.0320374.ref006","doi-asserted-by":"crossref","first-page":"5517","DOI":"10.3390\/ijms16035517","article-title":"Three-dimensional cell culture: a breakthrough in vivo","volume":"16","author":"D Antoni","year":"2015","journal-title":"Int J Mol Sci"},{"key":"pone.0320374.ref007","first-page":"765426","article-title":"Development and characterization of liposomal doxorubicin hydrochloride with palm oil","author":"B Sabeti","year":"2014","journal-title":"Biomed Res Int"},{"key":"pone.0320374.ref008","doi-asserted-by":"crossref","first-page":"975","DOI":"10.2147\/IJN.S68861","article-title":"Liposomes as nanomedical devices","volume":"10","author":"G Bozzuto","year":"2015","journal-title":"Int J Nanomedicine"},{"issue":"25","key":"pone.0320374.ref009","doi-asserted-by":"crossref","first-page":"3267","DOI":"10.2174\/092986709788803312","article-title":"Doxorubicin: the good, the bad and the ugly effect","volume":"16","author":"C Carvalho","year":"2009","journal-title":"Curr Med Chem"},{"issue":"12","key":"pone.0320374.ref010","doi-asserted-by":"crossref","first-page":"506","DOI":"10.3390\/bios11120506","article-title":"Construction of a Fibroblast-Associated Tumor Spheroid Model Based on a Collagen Drop Array Chip","volume":"11","author":"H Roh","year":"2021","journal-title":"Biosensors (Basel)"},{"issue":"53","key":"pone.0320374.ref011","doi-asserted-by":"crossref","first-page":"30976","DOI":"10.1039\/C9RA05636J","article-title":"Encapsulation of echinomycin in cyclodextrin inclusion complexes into liposomes: in vitro anti-proliferative and anti-invasive activity in glioblastoma","volume":"9","author":"W Alshaer","year":"2019","journal-title":"RSC Adv"},{"issue":"4","key":"pone.0320374.ref012","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1158\/1078-0432.CCR-23-0493","article-title":"Functional contribution and clinical implication of cancer-associated fibroblasts in glioblastoma","volume":"30","author":"PM Galbo","year":"2024","journal-title":"Clin Cancer Res"},{"issue":"27","key":"pone.0320374.ref013","doi-asserted-by":"crossref","first-page":"4833","DOI":"10.1016\/j.biomaterials.2009.05.043","article-title":"Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices","volume":"30","author":"KE Sung","year":"2009","journal-title":"Biomaterials"},{"issue":"2","key":"pone.0320374.ref014","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/labinvest.2008.126","article-title":"In vitro spheroid model of placental vasculogenesis: does it work?","volume":"89","author":"N Baal","year":"2009","journal-title":"Lab Invest"},{"issue":"4","key":"pone.0320374.ref015","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1038\/nrc2618","article-title":"Microenvironmental regulation of metastasis","volume":"9","author":"JA Joyce","year":"2009","journal-title":"Nat Rev Cancer"},{"issue":"Pt 13","key":"pone.0320374.ref016","first-page":"3015","article-title":"Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues","volume":"125","author":"BM Baker","year":"2012","journal-title":"J Cell Sci"},{"issue":"1","key":"pone.0320374.ref017","doi-asserted-by":"crossref","first-page":"R3","DOI":"10.1186\/bcr2218","article-title":"Novel multicellular organotypic models of normal and malignant breast: tools for dissecting the role of the microenvironment in breast cancer progression","volume":"11","author":"DL Holliday","year":"2009","journal-title":"Breast Cancer Res"},{"issue":"2","key":"pone.0320374.ref018","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1038\/sj.onc.1205053","article-title":"Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors","volume":"21","author":"ER Lawlor","year":"2002","journal-title":"Oncogene"},{"key":"pone.0320374.ref019","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.jphotobiol.2017.03.024","article-title":"Fluorescence properties of doxorubicin in PBS buffer and PVA films","volume":"170","author":"S Shah","year":"2017","journal-title":"J Photochem Photobiol B"},{"issue":"9","key":"pone.0320374.ref020","doi-asserted-by":"crossref","first-page":"e44947","DOI":"10.1371\/journal.pone.0044947","article-title":"Probing the dynamics of doxorubicin-DNA intercalation during the initial activation of apoptosis by fluorescence lifetime imaging microscopy (FLIM)","volume":"7","author":"N-T Chen","year":"2012","journal-title":"PLoS One"},{"issue":"3","key":"pone.0320374.ref021","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/S0301-4622(98)00150-1","article-title":"Deciphering the fluorescence signature of daunomycin and doxorubicin","volume":"73","author":"KK Karukstis","year":"1998","journal-title":"Biophys Chem"},{"issue":"9","key":"pone.0320374.ref022","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1111\/jphp.12257","article-title":"Comparison of pharmacokinetics, tissue distribution and pharmacodynamics of liposomal and free doxorubicin in tumour-bearing mice following intratumoral injection","volume":"66","author":"S Ren","year":"2014","journal-title":"J Pharm Pharmacol"},{"issue":"5","key":"pone.0320374.ref023","doi-asserted-by":"crossref","first-page":"419","DOI":"10.2165\/00003088-200342050-00002","article-title":"Pharmacokinetics of pegylated liposomal Doxorubicin: review of animal and human studies","volume":"42","author":"A Gabizon","year":"2003","journal-title":"Clin Pharmacokinet"},{"issue":"1\u20132","key":"pone.0320374.ref024","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.ijpharm.2018.07.019","article-title":"Direct quantification of unencapsulated doxorubicin in liposomal doxorubicin formulations using capillary electrophoresis","volume":"549","author":"SM Ansar","year":"2018","journal-title":"Int J Pharm"},{"issue":"5","key":"pone.0320374.ref025","doi-asserted-by":"crossref","first-page":"e92555","DOI":"10.1371\/journal.pone.0092555","article-title":"Therapeutic efficacy of combining pegylated liposomal doxorubicin and radiofrequency (RF) ablation: comparison between slow-drug-releasing, non-thermosensitive and fast-drug-releasing, thermosensitive nano-liposomes","volume":"9","author":"AV Andriyanov","year":"2014","journal-title":"PLoS One"},{"issue":"10","key":"pone.0320374.ref026","doi-asserted-by":"crossref","first-page":"1633","DOI":"10.1016\/j.bbamem.2006.05.028","article-title":"Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient","author":"A Fritze","year":"2006","journal-title":"Biochim Biophys Acta"},{"issue":"9","key":"pone.0320374.ref027","doi-asserted-by":"crossref","first-page":"1184","DOI":"10.1039\/c3ib40021b","article-title":"Microfluidic technique to measure intratumoral transport and calculate drug efficacy shows that binding is essential for doxorubicin and release hampers Doxil","volume":"5","author":"BJ Toley","year":"2013","journal-title":"Integr Biol (Camb)"},{"issue":"1","key":"pone.0320374.ref028","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1038\/mt.2009.160","article-title":"Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: cytotoxicity and mechanisms involved","volume":"18","author":"I Lentacker","year":"2010","journal-title":"Mol Ther"},{"issue":"8","key":"pone.0320374.ref029","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1016\/j.acthis.2012.01.006","article-title":"MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets","volume":"114","author":"JC Stockert","year":"2012","journal-title":"Acta Histochem"},{"issue":"3","key":"pone.0320374.ref030","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.cdp.2006.03.007","article-title":"Resveratrol modulation of signal transduction in apoptosis and cell survival: a mini-review","volume":"30","author":"S Fulda","year":"2006","journal-title":"Cancer Detect Prev"},{"key":"pone.0320374.ref031","doi-asserted-by":"crossref","first-page":"111708","DOI":"10.1016\/j.biopha.2021.111708","article-title":"Doxorubicin-induced cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for effective management","volume":"139","author":"PS Rawat","year":"2021","journal-title":"Biomed Pharmacother"},{"key":"pone.0320374.ref032","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.pharmthera.2016.03.013","article-title":"Three-dimensional culture systems in cancer research: Focus on tumor spheroid model","volume":"163","author":"S Nath","year":"2016","journal-title":"Pharmacol Ther"},{"issue":"5","key":"pone.0320374.ref033","doi-asserted-by":"crossref","first-page":"440","DOI":"10.4161\/cam.36224","article-title":"An introduction to the wound healing assay using live-cell microscopy","volume":"8","author":"JEN Jonkman","year":"2014","journal-title":"Cell Adh Migr"},{"issue":"11","key":"pone.0320374.ref034","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1038\/nrclinonc.2010.139","article-title":"Delivering nanomedicine to solid tumors","volume":"7","author":"RK Jain","year":"2010","journal-title":"Nat Rev Clin Oncol"},{"issue":"1","key":"pone.0320374.ref035","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1096\/fj.07-9150com","article-title":"Enhanced macromolecule diffusion deep in tumors after enzymatic digestion of extracellular matrix collagen and its associated proteoglycan decorin","volume":"22","author":"M Magzoub","year":"2008","journal-title":"FASEB J"},{"issue":"5","key":"pone.0320374.ref036","doi-asserted-by":"crossref","first-page":"1371","DOI":"10.1021\/acs.bioconjchem.9b00136","article-title":"Penetration and uptake of nanoparticles in 3D tumor spheroids","volume":"30","author":"A Tchoryk","year":"2019","journal-title":"Bioconjug Chem"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0320374","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,3,25]],"date-time":"2025-03-25T19:26:35Z","timestamp":1742930795000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0320374"}},"subtitle":[],"editor":[{"given":"B. H. Jaswanth","family":"Gowda","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,3,25]]},"references-count":36,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2025,3,25]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0320374","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,25]]}}}