{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T17:13:17Z","timestamp":1779297197737,"version":"3.51.4"},"reference-count":44,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T00:00:00Z","timestamp":1779148800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"CSL Vifor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: The blood\u2013brain barrier (BBB) presents a major challenge for delivering therapeutics to the central nervous system (CNS) due to its highly selective permeability. Human brain microvascular endothelial cells (hBMECs), the principal cellular component of the BBB, tightly regulate molecular transport and restrict the entry of many CNS-targeted therapies. Lipid-based nanoparticles have emerged as promising carriers for BBB transport because of their biocompatibility, tunable surface properties, and cargo encapsulation capabilities. One strategy to enhance nanoparticle transport involves surface functionalization with ligands that exploit endogenous transcytosis pathways. Mannose-6-phosphate (M6P), a glycan implicated in the brain entry of certain proteins and viruses, represents a potential targeting ligand for this purpose. Methods: In this study, we established a physiologically relevant in vitro BBB model using human-induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs) to evaluate M6P-functionalized liposomes for BBB transport. Fluorophore-labeled liposomes were used to monitor nanoparticle uptake and transcytosis. Results: M6P-functionalized liposomes exhibited significantly enhanced uptake in hiPSC-BMECs compared with non-functionalized control liposomes. Pharmacological inhibition studies supported the involvement of a clathrin-sensitive endocytic pathway. Transcytosis assays demonstrated enhanced BBB crossing of M6P-functionalized liposomes, with transport increasing according to ligand density and reaching approximately 55% of the transport observed for transferrin under the same experimental conditions. Following transcytosis, intact M6P-functionalized liposomes showed significantly higher uptake by downstream hiPSC-derived neurons and astrocytoma cells compared with control formulations. Conclusions: Together, these findings support M6P-functionalization as a promising strategy to enhance liposome uptake and transcytosis across a human-relevant in vitro BBB model. This work provides a proof-of-concept framework for the development and optimization of glycan-functionalized nanocarriers for CNS-directed delivery.<\/jats:p>","DOI":"10.3390\/pharmaceutics18050619","type":"journal-article","created":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T16:39:34Z","timestamp":1779295174000},"page":"619","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mannose-6-Phosphate-Tagged Liposomes Exhibit Increased Transcytosis Across Human Blood\u2013Brain Barrier Model"],"prefix":"10.3390","volume":"18","author":[{"given":"Margarita C.","family":"Dinamarca","sequence":"first","affiliation":[{"name":"Nanopharmaceutical and Regulatory Science Group, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-8536-4302","authenticated-orcid":false,"given":"Boris","family":"Sevarika","sequence":"additional","affiliation":[{"name":"Nanopharmaceutical and Regulatory Science Group, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7575-5198","authenticated-orcid":false,"given":"Scott","family":"McNeil","sequence":"additional","affiliation":[{"name":"Nanopharmaceutical and Regulatory Science Group, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1038\/nrn1824","article-title":"Astrocyte\u2013endothelial interactions at the blood\u2013brain barrier","volume":"7","author":"Abbott","year":"2006","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1602\/neurorx.2.1.3","article-title":"The blood-brain barrier: Bottleneck in brain drug development","volume":"2","author":"Pardridge","year":"2005","journal-title":"NeuroRX"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1089\/nat.2018.0721","article-title":"Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility","volume":"28","author":"Kulkarni","year":"2018","journal-title":"Nucleic Acid Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1038\/s41565-021-00898-0","article-title":"The current landscape of nucleic acid therapeutics","volume":"16","author":"Kulkarni","year":"2021","journal-title":"Nat. Nanotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Sercombe, L., Veerati, T., Moheimani, F., Wu, S.Y., Sood, A.K., and Hua, S. (2015). Advances and Challenges of Liposome Assisted Drug Delivery. Front. Pharmacol., 6.","DOI":"10.3389\/fphar.2015.00286"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1038\/s41583-020-0322-2","article-title":"Neuronal regulation of the blood\u2013brain barrier and neurovascular coupling","volume":"21","author":"Kaplan","year":"2020","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"233","DOI":"10.3892\/etm.2014.1727","article-title":"Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants","volume":"211","author":"Yu","year":"2014","journal-title":"J. Exp. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"eads3204","DOI":"10.1126\/science.ads3204","article-title":"Transferrin receptor\u2013targeted anti-amyloid antibody enhances brain delivery and mitigates ARIA","volume":"389","author":"Pizzo","year":"2025","journal-title":"Science"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"10396","DOI":"10.1038\/s41598-017-11220-1","article-title":"Targeting transferrin receptors at the blood-brain barrier improves the uptake of immunoliposomes and subsequent cargo transport into the brain parenchyma","volume":"7","author":"Johnsen","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"101665","DOI":"10.1016\/j.pneurobio.2019.101665","article-title":"Targeting the transferrin receptor for brain drug delivery","volume":"181","author":"Johnsen","year":"2019","journal-title":"Prog. Neurobiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"8662","DOI":"10.1073\/pnas.1307152110","article-title":"Transcytosis and brain uptake of transferrin-containing nanoparticles by tuning avidity to transferrin receptor","volume":"110","author":"Wiley","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2057","DOI":"10.1038\/s41467-023-37632-4","article-title":"Targeting neuronal lysosomal dysfunction caused by \u03b2-glucocerebrosidase deficiency with an enzyme-based brain shuttle construct","volume":"14","author":"Gehrlein","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"116038","DOI":"10.1016\/j.celrep.2025.116038","article-title":"Dual targeting of transferrin receptor and CD98hc enhances brain exposure of large molecules","volume":"44","author":"Wells","year":"2025","journal-title":"Cell Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"26875","DOI":"10.1038\/s41598-024-75300-9","article-title":"Lysosomal enzyme binding to the cation-independent mannose 6-phosphate receptor is regulated allosterically by insulin-like growth factor 2","volume":"14","author":"Bohnsack","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1091\/mbc.e03-07-0497","article-title":"Endocytosed Cation-Independent Mannose 6-Phosphate Receptor Traffics via the Endocytic Recycling Compartment en Route to thetrans-Golgi Network and a Subpopulation of Late Endosomes","volume":"15","author":"Lin","year":"2004","journal-title":"Mol. Biol. Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"6197","DOI":"10.1128\/JVI.76.12.6197-6204.2002","article-title":"Cation-Independent Mannose 6-Phosphate Receptor Blocks Apoptosis Induced by Herpes Simplex Virus 1 Mutants Lacking Glycoprotein D and Is Likely the Target of Antiapoptotic Activity of the Glycoprotein","volume":"76","author":"Zhou","year":"2002","journal-title":"J. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.virusres.2017.12.002","article-title":"Most rotavirus strains require the cation-independent mannose-6-phosphate receptor, sortilin-1, and cathepsins to enter cells","volume":"245","author":"Casorla","year":"2018","journal-title":"Virus Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1503","DOI":"10.1038\/sj.ki.5000265","article-title":"Renin\/prorenin receptors","volume":"69","author":"Nguyen","year":"2006","journal-title":"Kidney Int."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1093\/glycob\/4.1.13","article-title":"Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate","volume":"4","author":"Mbemba","year":"1994","journal-title":"Glycobiology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1038\/nbt.2247","article-title":"Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells","volume":"30","author":"Lippmann","year":"2012","journal-title":"Nat. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/s12035-017-0728-7","article-title":"Intranasal Administration of TAT-Conjugated Lipid Nanocarriers Loading GDNF for Parkinson\u2019s Disease","volume":"55","author":"Hernando","year":"2018","journal-title":"Mol. Neurobiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1073\/pnas.0805165106","article-title":"Wnt\/\u03b2-catenin signaling is required for CNS, but not non-CNS, angiogenesis","volume":"106","author":"Daneman","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e70992","DOI":"10.7554\/eLife.70992","article-title":"Wnt signaling mediates acquisition of blood-brain barrier properties in na\u00efve endothelium derived from human pluripotent stem cells","volume":"10","author":"Gastfriend","year":"2021","journal-title":"eLife"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1083\/jcb.200810040","article-title":"Formation of the blood\u2013brain barrier: Wnt signaling seals the deal","volume":"183","author":"Polakis","year":"2008","journal-title":"J. Cell Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"114665","DOI":"10.1016\/j.ejpb.2025.114665","article-title":"Mannose-6-phosphate-PEG-lipid conjugates improve liposomal uptake","volume":"209","author":"Sevarika","year":"2025","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1799","DOI":"10.1038\/s41598-018-20064-2","article-title":"Preferential amplification of a human mitochondrial DNA deletion in vitro and in vivo","volume":"8","author":"Russell","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1038\/nature06968","article-title":"The ground state of embryonic stem cell self-renewal","volume":"453","author":"Ying","year":"2008","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1042\/BJ20070797","article-title":"The selectivity of protein kinase inhibitors: A further update","volume":"408","author":"Bain","year":"2007","journal-title":"Biochem. J."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e122","DOI":"10.1002\/cpsc.122","article-title":"Human iPSC-Derived Blood-Brain Barrier Models: Valuable Tools for Preclinical Drug Discovery and Development?","volume":"55","author":"Oerter","year":"2020","journal-title":"Curr. Protoc. Stem Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1380","DOI":"10.1016\/j.stemcr.2019.05.008","article-title":"A Simplified, Fully Defined Differentiation Scheme for Producing Blood-Brain Barrier Endothelial Cells from Human iPSCs","volume":"12","author":"Neal","year":"2019","journal-title":"Stem Cell Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"13957","DOI":"10.1038\/s41598-019-50193-1","article-title":"Tissue-engineered blood-brain barrier models via directed differentiation of human induced pluripotent stem cells","volume":"9","author":"Grifno","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1016\/j.stemcr.2017.02.021","article-title":"Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells","volume":"8","author":"Cubukova","year":"2017","journal-title":"Stem Cell Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/s12987-017-0059-0","article-title":"Accelerated differentiation of human induced pluripotent stem cells to blood\u2013brain barrier endothelial cells","volume":"14","author":"Hollmann","year":"2017","journal-title":"Fluids Barriers CNS"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1007\/s12975-010-0049-x","article-title":"High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)\u2013Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations","volume":"2","author":"Hoffmann","year":"2011","journal-title":"Transl. Stroke Res."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Haqqani, A.S., B\u00e9langer, K., and Stanimirovic, D.B. (2024). Receptor-mediated transcytosis for brain delivery of therapeutics: Receptor classes and criteria. Front. Drug Deliv., 4.","DOI":"10.3389\/fddev.2024.1360302"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Guo, Z. (2023). Ganglioside GM1 and the Central Nervous System. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24119558"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1111\/j.1574-6968.2006.00545.x","article-title":"Rafting with cholera toxin: Endocytosis and trafficking from plasma membrane to ER","volume":"266","author":"Chinnapen","year":"2007","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Dutta, D., Williamson, C.D., Cole, N.B., and Donaldson, J.G. (2012). Pitstop 2 Is a Potent Inhibitor of Clathrin-Independent Endocytosis. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0045799"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1182","DOI":"10.1111\/tra.12100","article-title":"Sialic Acid Linkage in Glycosphingolipids Is a Molecular Correlate for Trafficking and Delivery of Extracellular Cargo","volume":"14","author":"Ravindran","year":"2013","journal-title":"Traffic"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3254","DOI":"10.1091\/mbc.e02-12-0809","article-title":"Selective Caveolin-1\u2013dependent Endocytosis of Glycosphingolipids","volume":"14","author":"Singh","year":"2003","journal-title":"Mol. Biol. Cell"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2767","DOI":"10.1038\/s41467-024-47109-7","article-title":"Adhesion energy controls lipid binding-mediated endocytosis","volume":"15","author":"Groza","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1242\/jcs.030338","article-title":"Binding of laminin-1 to monosialoganglioside GM1 in lipid rafts is crucial for neurite outgrowth","volume":"122","author":"Ichikawa","year":"2009","journal-title":"J. Cell Sci."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3124","DOI":"10.1002\/1873-3468.14532","article-title":"Regulation of signal transduction by gangliosides in lipid rafts: Focus on GM3\u2013IR and GM1\u2013TrkA interactions","volume":"596","author":"Lunghi","year":"2022","journal-title":"FEBS Lett."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1023\/A:1006948027674","article-title":"Transferrin and Transferrin Receptor Function in Brain Barrier Systems","volume":"20","author":"Moos","year":"2000","journal-title":"Cell. Mol. Neurobiol."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/18\/5\/619\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T16:43:03Z","timestamp":1779295383000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/18\/5\/619"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,19]]},"references-count":44,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["pharmaceutics18050619"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics18050619","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,19]]}}}