{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:59:16Z","timestamp":1785437956147,"version":"3.56.0"},"reference-count":34,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T00:00:00Z","timestamp":1778630400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"TERUMO Life Science Foundation","award":["24-III3017"],"award-info":[{"award-number":["24-III3017"]}]},{"DOI":"10.13039\/501100004330","name":"Smoking Research Foundation","doi-asserted-by":"publisher","award":["2024G012"],"award-info":[{"award-number":["2024G012"]}],"id":[{"id":"10.13039\/501100004330","id-type":"DOI","asserted-by":"publisher"}]},{"award":["2024G012"],"award-info":[{"award-number":["2024G012"]}],"id":[{"id":"https:\/\/ror.org\/04q4hbm31","id-type":"ROR","asserted-by":"publisher"}]},{"name":"The Science Research Promotion Fund"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Controlled delivery using nanoparticle-based systems has attracted considerable attention; however, achieving cell-type specificity remains a major challenge. To address this issue, we focused on the intrinsic cell tropism of viruses. The hepatocyte tropism of hepatitis B virus (HBV) is mediated by interactions between its large envelope protein (L protein) and host factors, including the sodium taurocholate cotransporting polypeptide (NTCP). In this study, we explored viral-like secretory vesicles (VLSVs) displaying HBV spike proteins as a virus-inspired vesicle platform for hepatocyte targeting. We previously established a method for producing VLSVs from HBV L- and S-expressing HEK293T cells. In the present study, we developed an improved protocol using exosome-depleted fetal calf serum and optimized ultracentrifugation, resulting in VLSVs with comparable particle numbers and sizes but approximately tenfold higher protein content per particle. VLSVs were concentrated using a two-layer sucrose cushion, labeled with DiI, and purified by sucrose density gradient ultracentrifugation. We evaluated DiI uptake in hepatocyte-derived cells (HepG2 and Huh7), non-hepatic cells (MDA-MB231, H1299, HeLa, and Vero), and NTCP-overexpressing HepG2 cells. VLSVs showed preferential uptake in the following order: NTCP-overexpressing HepG2 &gt; HepG2 &gt; Huh7 &gt; non-hepatic cells. Furthermore, removal of the N-terminal Flag tag from the L protein enhanced hepatocyte-associated uptake, suggesting the importance of preserving the native structure of the preS1 domain. While vesicle characterization and mechanistic validation remain to be further investigated, these findings provide a proof-of-concept for a virus-inspired vesicle platform exhibiting preferential uptake in hepatocyte-derived cells.<\/jats:p>","DOI":"10.3390\/ijms27104331","type":"journal-article","created":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T07:44:26Z","timestamp":1778658266000},"page":"4331","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["HBV Envelope Protein-Bearing Vesicles Show Preferential Uptake in Hepatocyte-Derived Cells"],"prefix":"10.3390","volume":"27","author":[{"given":"Eri","family":"Takayama","sequence":"first","affiliation":[{"name":"Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Misaki","family":"Enomoto","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manami","family":"Nagae","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Momoko","family":"Tomoda","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuta","family":"Miyazumi","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0202-4009","authenticated-orcid":false,"given":"Yuki","family":"Iwaisako","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryota","family":"Shirasawa","sequence":"additional","affiliation":[{"name":"Analytical Technology Division, HORIBA Techno Service Co., Ltd., Kyoto 601-8305, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3554-0755","authenticated-orcid":false,"given":"Youichi","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka 569-8686, Japan"},{"name":"Laboratory of Biosafety Research, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka 569-8686, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Nakano","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka 569-8686, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keiji","family":"Ueda","sequence":"additional","affiliation":[{"name":"Division of Virology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5165-0789","authenticated-orcid":false,"given":"Masahiro","family":"Fujimuro","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-4939-9798-5_1","article-title":"An Overview of Drug Delivery Systems","volume":"2059","author":"Jain","year":"2020","journal-title":"Methods Mol. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1080\/10717544.2020.1748758","article-title":"Prospects and Challenges of Extracellular Vesicle-Based Drug Delivery System: Considering Cell Source","volume":"27","author":"Meng","year":"2020","journal-title":"Drug Deliv."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1016\/j.apsb.2019.08.003","article-title":"Recent Progress in Drug Delivery","volume":"9","author":"Li","year":"2019","journal-title":"Acta Pharm. Sin. B"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1016\/j.addr.2011.10.007","article-title":"Nanotechnological Advances for the Delivery of CNS Therapeutics","volume":"64","author":"Wong","year":"2012","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_5","unstructured":"Howley, P.M., and Knipe, D.M. (2022). Hepadnaviridae. Fields Virology, Wolters Kluwer. [7th ed.]."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1146\/annurev.bi.56.070187.003251","article-title":"The Molecular Biology of the Hepatitis B Viruses","volume":"56","author":"Ganem","year":"1987","journal-title":"Annu. Rev. Biochem."},{"key":"ref_7","first-page":"61","article-title":"Assembly of Hepadnaviral Virions and Subviral Particles","volume":"168","author":"Ganem","year":"1991","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1128\/jvi.52.2.396-402.1984","article-title":"Large Surface Proteins of Hepatitis B Virus Containing the pre-S Sequence","volume":"52","author":"Heermann","year":"1984","journal-title":"J. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"9511","DOI":"10.1128\/JVI.77.17.9511-9521.2003","article-title":"Pre-S1 Antigen-Dependent Infection of Tupaia Hepatocyte Cultures with Human Hepatitis B Virus","volume":"77","author":"Glebe","year":"2003","journal-title":"J. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1613","DOI":"10.1128\/JVI.79.3.1613-1622.2005","article-title":"Efficient Inhibition of Hepatitis B Virus Infection by Acylated Peptides Derived from the Large Viral Surface Protein","volume":"79","author":"Gripon","year":"2005","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e00049","DOI":"10.7554\/eLife.00049","article-title":"Sodium Taurocholate Cotransporting Polypeptide Is a Functional Receptor for Human Hepatitis B and D Virus","volume":"1","author":"Yan","year":"2012","journal-title":"eLife"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1070","DOI":"10.1053\/j.gastro.2013.12.024","article-title":"Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-Transporting Polypeptide for Species-Specific Entry into Hepatocytes","volume":"146","author":"Ni","year":"2014","journal-title":"Gastroenterology"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Herrscher, C., Roingeard, P., and Blanchard, E. (2020). Hepatitis B Virus Entry into Cells. Cells, 9.","DOI":"10.3390\/cells9061486"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1038\/s41586-022-04857-0","article-title":"Structural Insights into the HBV Receptor and Bile Acid Transporter NTCP","volume":"606","author":"Park","year":"2022","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1097\/00000441-197509000-00025","article-title":"Purified and Inactivated Human Hepatitis B Vaccine: Progress Report","volume":"270","author":"Hilleman","year":"1975","journal-title":"Am. J. Med. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1097\/00000441-197509000-00024","article-title":"Hepatitis B Subunit Vaccine: A Preliminary Report of Safety and Efficacy Tests in Chimpanzees","volume":"270","author":"Purcell","year":"1975","journal-title":"Am. J. Med. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/S0163-4453(86)92563-6","article-title":"Production and Immunological Analysis of Recombinant Hepatitis B Vaccine","volume":"13","author":"Emini","year":"1986","journal-title":"J. Infect."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"S69\u2013S73; discussion S79\u2013S80","DOI":"10.1016\/0264-410X(90)90221-7","article-title":"Development and Production Aspects of a Recombinant Yeast-Derived Hepatitis B Vaccine","volume":"8","author":"Stephenne","year":"1990","journal-title":"Vaccine"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"12642","DOI":"10.1021\/acsnano.9b08756","article-title":"Modular Hepatitis B Virus-like Particle Platform for Biosensing and Drug Delivery","volume":"14","author":"Hartzell","year":"2020","journal-title":"ACS Nano"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1038\/nbt843","article-title":"Nanoparticles for the Delivery of Genes and Drugs to Human Hepatocytes","volume":"21","author":"Yamada","year":"2003","journal-title":"Nat. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3521","DOI":"10.1128\/jvi.65.7.3521-3529.1991","article-title":"Three Envelope Proteins of Hepatitis B Virus: Large S, Middle S, and Major S Proteins Needed for the Formation of Dane Particles","volume":"65","author":"Ueda","year":"1991","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.bbrc.2021.10.009","article-title":"Human Hepatitis B Virus-Derived Virus-Like Particle as a Drug and DNA Delivery Carrier","volume":"581","author":"Sakai","year":"2021","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1016\/j.jhep.2016.10.009","article-title":"Human Stem Cell-Derived Hepatocytes as a Model for Hepatitis B Virus Infection, Spreading and Virus\u2013Host Interactions","volume":"66","author":"Xia","year":"2017","journal-title":"J. Hepatol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1016\/j.bbrc.2013.12.052","article-title":"Evaluation and Identification of Hepatitis B Virus Entry Inhibitors Using HepG2 Cells Overexpressing a Membrane Transporter NTCP","volume":"443","author":"Iwamoto","year":"2014","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_25","first-page":"122","article-title":"Role of Glycosaminoglycans for Binding and Infection of Hepatitis B Virus","volume":"10","author":"Leistner","year":"2008","journal-title":"Cell Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1759","DOI":"10.1002\/hep.21896","article-title":"Hepatitis B Virus Infection Initiates with a Large Surface Protein-Dependent Binding to Heparan Sulfate Proteoglycans","volume":"46","author":"Schulze","year":"2007","journal-title":"Hepatology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1038\/s41594-023-01191-5","article-title":"Structural Basis of Hepatitis B Virus Receptor Binding","volume":"31","author":"Asami","year":"2024","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"8487","DOI":"10.1073\/pnas.1811064116","article-title":"Epidermal Growth Factor Receptor Is a Host-Entry Cofactor Triggering Hepatitis B Virus Internalization","volume":"116","author":"Iwamoto","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Hu, Q., Zhang, F., Duan, L., Wang, B., Ye, Y., Li, P., Li, D., Yang, S., Zhou, L., and Chen, W. (2020). E-Cadherin Plays a Role in Hepatitis B Virus Entry Through Affecting Glycosylated Sodium-Taurocholate Cotransporting Polypeptide Distribution. Front. Cell. Infect. Microbiol., 10.","DOI":"10.3389\/fcimb.2020.00074"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"723","DOI":"10.1016\/j.jhep.2013.11.022","article-title":"Cyclosporin A Inhibits Hepatitis B and Hepatitis D Virus Entry by Cyclophilin-Independent Interference with the NTCP Receptor","volume":"60","author":"Nkongolo","year":"2014","journal-title":"J. Hepatol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1016\/j.jhep.2014.05.018","article-title":"Kinetics of the Bile Acid Transporter and Hepatitis B Virus Receptor Na+\/Taurocholate Cotransporting Polypeptide (NTCP) in Hepatocytes","volume":"61","author":"Mohr","year":"2014","journal-title":"J. Hepatol."},{"key":"ref_32","first-page":"2745","article-title":"High-Efficiency Transformation of Mammalian Cells by Plasmid DNA","volume":"7","author":"Chen","year":"1987","journal-title":"Mol. Cell. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"6671","DOI":"10.1038\/s41598-020-63438-1","article-title":"MG132 Exerts Anti-Viral Activity against HSV-1 by Overcoming Virus-Mediated Suppression of the ERK Signaling Pathway","volume":"10","author":"Ishimaru","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e00475-23","DOI":"10.1128\/jvi.00475-23","article-title":"Kaposi\u2019s Sarcoma-Associated Herpesvirus ORF67.5 Functions as a Component of the Terminase Complex","volume":"97","author":"Iwaisako","year":"2023","journal-title":"J. Virol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/10\/4331\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T07:46:11Z","timestamp":1778658371000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/10\/4331"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,13]]},"references-count":34,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["ijms27104331"],"URL":"https:\/\/doi.org\/10.3390\/ijms27104331","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,13]]}}}