{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:50:27Z","timestamp":1760151027653,"version":"build-2065373602"},"reference-count":52,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2022,2,15]],"date-time":"2022-02-15T00:00:00Z","timestamp":1644883200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Protein-based carriers are promising vehicles for the intracellular delivery of therapeutics. In this study, we designed and studied adenovirus protein fiber constructs with potential applications as carriers for the delivery of protein and nanoparticle cargoes. We used as a basic structural framework the fibrous shaft segment of the adenovirus fiber protein comprising of residues 61\u2013392, connected to the fibritin foldon trimerization motif at the C-terminal end. A fourteen-amino-acid biotinylation sequence was inserted immediately after the N-terminal, His-tagged end of the construct in order to enable the attachment of a biotin moiety in vivo. We report herein that this His-tag biotinylated construct folds into thermally and protease-stable fibrous nanorods that can be internalized into cells and are not cytotoxic. Moreover, they can bind to proteins and nanoparticles through the biotin\u2013streptavidin interaction and mediate their delivery to cells. We demonstrate that streptavidin-conjugated gold nanoparticles can be transported into NIH3T3 fibroblast and HeLa cancer cell lines. Furthermore, two streptavidin-conjugated model proteins, alkaline phosphatase and horseradish peroxidase can be delivered into the cell cytoplasm in their enzymatically active form. This work is aimed at establishing the proof-of-principle for the rational engineering of diverse functionalities onto the initial protein structural framework and the use of adenovirus fiber-based proteins as nanorods for the delivery of nanoparticles and model proteins. These constructs could constitute a stepping stone for the development of multifunctional and modular fibrous nanorod platforms that can be tailored to applications at the sequence level.<\/jats:p>","DOI":"10.3390\/biom12020308","type":"journal-article","created":{"date-parts":[[2022,2,15]],"date-time":"2022-02-15T22:43:22Z","timestamp":1644965002000},"page":"308","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Adenovirus Fibers as Ultra-Stable Vehicles for Intracellular Nanoparticle and Protein Delivery"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4825-9022","authenticated-orcid":false,"given":"Chrysoula","family":"Kokotidou","sequence":"first","affiliation":[{"name":"Department of Materials Science and Technology, University of Crete, 70013 Heraklion, Crete, Greece"},{"name":"Institute of Electronic Structure and Laser (IESL), FORTH, 70013 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fani","family":"Tsitouroudi","sequence":"additional","affiliation":[{"name":"Institute of Electronic Structure and Laser (IESL), FORTH, 70013 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Georgios","family":"Nistikakis","sequence":"additional","affiliation":[{"name":"Department of Materials Science and Technology, University of Crete, 70013 Heraklion, Crete, Greece"},{"name":"Institute of Electronic Structure and Laser (IESL), FORTH, 70013 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marita","family":"Vasila","sequence":"additional","affiliation":[{"name":"Department of Materials Science and Technology, University of Crete, 70013 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8889-8606","authenticated-orcid":false,"given":"Katerina","family":"Papanikolopoulou","sequence":"additional","affiliation":[{"name":"Department of Materials Science and Technology, University of Crete, 70013 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2949-1619","authenticated-orcid":false,"given":"Androniki","family":"Kretsovali","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology and Biotechnology (IMBB), FORTH, 70013 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna","family":"Mitraki","sequence":"additional","affiliation":[{"name":"Department of Materials Science and Technology, University of Crete, 70013 Heraklion, Crete, Greece"},{"name":"Institute of Electronic Structure and Laser (IESL), FORTH, 70013 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"941","DOI":"10.2217\/nnm-2016-0393","article-title":"Intracellular delivery of proteins by nanocarriers","volume":"12","author":"Ray","year":"2017","journal-title":"Nanomedicine"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/B978-0-12-398342-8.00002-1","article-title":"Adenovirus Strategies for Tissue-Specific Targeting","volume":"Volume 115","author":"Curiel","year":"2012","journal-title":"Applications of Viruses for Cancer Therapy"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"830","DOI":"10.1038\/sj.cgt.7700928","article-title":"Transductional targeting of adenovirus vectors for gene therapy","volume":"13","author":"Glasgow","year":"2006","journal-title":"Cancer Gene Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.gendis.2017.04.001","article-title":"Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine","volume":"4","author":"Lee","year":"2017","journal-title":"Genes Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1038\/s41417-017-0002-1","article-title":"Development of an adenovirus vector vaccine platform for targeting dendritic cells","volume":"25","author":"Sharma","year":"2018","journal-title":"Cancer Gene Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1038\/s41541-021-00356-x","article-title":"Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic","volume":"6","author":"Lorincz","year":"2021","journal-title":"npj Vaccines"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1126\/science.1187292","article-title":"Crystal Structure of Human Adenovirus at 3.5 angstrom Resolution","volume":"329","author":"Reddy","year":"2010","journal-title":"Science"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1038\/nbt0197-52","article-title":"Adenovirus dodecahedron, a new vector for human gene transfer","volume":"15","author":"Fender","year":"1997","journal-title":"Nat. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Zochowska, M., Paca, A., Schoehn, G., Andrieu, J.P., Chroboczek, J., Dublet, B., and Szolajska, E. (2009). Adenovirus Dodecahedron, as a Drug Delivery Vector. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0005569"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4960","DOI":"10.1128\/JVI.77.8.4960-4964.2003","article-title":"Adenovirus dodecahedron allows large multimeric protein transduction in human cells","volume":"77","author":"Fender","year":"2003","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Besson, S., Vragniau, C., Vassal-Stermann, E., Dagher, M.C., and Fender, P. (2020). The Adenovirus Dodecahedron: Beyond the Platonic Story. Viruses, 12.","DOI":"10.3390\/v12070718"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"795","DOI":"10.1038\/sj.gt.3301448","article-title":"3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins","volume":"8","author":"Kasahara","year":"2001","journal-title":"Gene Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1038\/sj.gt.3302729","article-title":"Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: Implications for gene transfer","volume":"13","author":"Rentsendorj","year":"2006","journal-title":"Gene Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1007\/s11095-005-9225-1","article-title":"Specific delivery of corroles to cells via noncovalent conjugates with viral proteins","volume":"23","author":"Agadjanian","year":"2006","journal-title":"Pharm. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1006\/mthe.2001.0472","article-title":"Adenovirus hexon protein enhances nuclear delivery and increases transgene expression of polyethylenimine\/plasmid DNA vectors","volume":"4","author":"Carlisle","year":"2001","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1038\/sj.gt.3302402","article-title":"The Ad5 fiber mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel cell entry pathway","volume":"12","author":"Rentsendorj","year":"2005","journal-title":"Gene Ther."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1016\/S0022-2836(05)80170-6","article-title":"Structure of adenovirus fibre. II. Morphology of single fibres","volume":"215","author":"Ruigrok","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1006\/viro.1993.1241","article-title":"The short tail-fiber of bacteriophage T4: Molecular structure and a mechanism for its conformational transition","volume":"194","author":"Makhov","year":"1993","journal-title":"Virology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1006\/jsbi.2002.4447","article-title":"Review: Conformation and folding of novel Beta-structural elements in viral fiber proteins: The triple Beta-spiral and triple Beta-helix","volume":"137","author":"Mitraki","year":"2002","journal-title":"J. Struct. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/0042-6822(91)90784-9","article-title":"Deletion analysis of functional domains in baculovirus-expressed adenovirus type 2 fiber","volume":"185","author":"Novelli","year":"1991","journal-title":"Virology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"7071","DOI":"10.1128\/jvi.70.10.7071-7078.1996","article-title":"Domains required for assembly of adenovirus type 2 fiber trimers","volume":"70","author":"Hong","year":"1996","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1046\/j.1432-1327.1999.00683.x","article-title":"Unfolding studies of human adenovirus type 2 fibre trimers Evidence for a stable domain","volume":"264","author":"Mitraki","year":"1999","journal-title":"Eur. J. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"8150","DOI":"10.1021\/la701042t","article-title":"Multiscale surface self-assembly of an amyloid-like peptide","volume":"23","author":"Lepere","year":"2007","journal-title":"Langmuir"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/S0014-5793(00)01184-4","article-title":"A peptide from the adenovirus fiber shaft forms amyloid-type fibrils","volume":"468","author":"Luckey","year":"2000","journal-title":"FEBS Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2481","DOI":"10.1074\/jbc.M406282200","article-title":"Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber","volume":"280","author":"Papanikolopoulou","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_26","first-page":"97","article-title":"Natural triple beta-stranded fibrous folds","volume":"73","author":"Mitraki","year":"2006","journal-title":"Fibrous Proteins: Amyloids Prions Beta Proteins"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1038\/44880","article-title":"A triple beta-spiral in the adenovirus fibre shaft reveals a new structural motif for a fibrous protein","volume":"401","author":"Mitraki","year":"1999","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1006\/jmbi.1998.2214","article-title":"Crystal structure of GCN4-pI(Q)I, a trimeric coiled coil with buried polar residues","volume":"284","author":"Eckert","year":"1998","journal-title":"J. Mol. Biol."},{"key":"ref_29","first-page":"817","article-title":"The carboxy-terminal domain initiates trimerization of bacteriophage T4 fibritin","volume":"64","author":"Letarov","year":"1999","journal-title":"Biochemistry (Moscow)"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1940","DOI":"10.1038\/sj.gt.3301323","article-title":"Recombinant adenovirus vectors with knobless fibers for targeted gene transfer","volume":"7","author":"Haisma","year":"2000","journal-title":"Gene Ther."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"627","DOI":"10.1038\/sj.gt.3302916","article-title":"Targeting EGFR with metabolically biotinylated fiber-mosaic adenovirus","volume":"14","author":"Pereboeva","year":"2007","journal-title":"Gene Ther."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"8991","DOI":"10.1074\/jbc.M311791200","article-title":"Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin","volume":"279","author":"Papanikolopoulou","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.jmb.2004.07.008","article-title":"Adenovirus fibre shaft sequences fold into the native triple beta-spiral fold when N-terminally fused to the bacteriophage T4 fibritin foldon trimerisation motif","volume":"342","author":"Papanikolopoulou","year":"2004","journal-title":"J. Mol. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1629","DOI":"10.1007\/s10529-012-0942-3","article-title":"Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin","volume":"34","author":"Li","year":"2012","journal-title":"Biotechnol. Lett."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Predonzani, A., Arnoldi, F., L\u00f3pez-Requena, A., and Burrone, O.R. (2008). In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system. BMC Biotechnol., 8.","DOI":"10.1186\/1472-6750-8-41"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Evans, R., O\u2019Neill, M., Pritzel, A., Antropova, N., Senior, A., Green, T., \u017d\u00eddek, A., Bates, R., Blackwell, S., and Yim, J. (2021). Protein complex prediction with AlphaFold-Multimer. bioRxiv.","DOI":"10.1101\/2021.10.04.463034"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/1475-2859-4-27","article-title":"Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins","volume":"4","author":"Morell","year":"2005","journal-title":"Microb. Cell Factories"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.tibtech.2006.02.007","article-title":"Protein quality in bacterial inclusion bodies","volume":"24","author":"Ventura","year":"2006","journal-title":"Trends Biotechnol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/S0169-409X(01)00221-6","article-title":"Histidine-rich peptides and polymers for nucleic acids delivery","volume":"53","author":"Pichon","year":"2001","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/j.nano.2013.11.006","article-title":"Sheltering DNA in self-organizing, protein-only nano-shells as artificial viruses for gene delivery","volume":"10","author":"Unzueta","year":"2014","journal-title":"Nanomedicine"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1186\/1475-2859-10-101","article-title":"Biological activities of histidine-rich peptides; merging biotechnology and nanomedicine","volume":"10","author":"Corchero","year":"2011","journal-title":"Microb. Cell Factories"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"259","DOI":"10.2217\/nnm.09.98","article-title":"Protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (R9) peptide","volume":"5","author":"Vazquez","year":"2010","journal-title":"Nanomedicine"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1002\/ijch.201400097","article-title":"Protein Needles as Molecular Templates for Artificial Metalloenzymes","volume":"55","author":"Inaba","year":"2015","journal-title":"Isr. J. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3111","DOI":"10.1039\/C5MB00327J","article-title":"A metal carbonyl-protein needle composite designed for intracellular CO delivery to modulate NF-kappa B activity","volume":"11","author":"Inaba","year":"2015","journal-title":"Mol. Biosyst."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1505","DOI":"10.1246\/cl.140481","article-title":"Intracellular Protein Delivery System with Protein Needle-GFP Construct","volume":"43","author":"Inaba","year":"2014","journal-title":"Chem. Lett."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"4302","DOI":"10.1021\/acs.biomac.0c01103","article-title":"Multifunctional Amyloid Oligomeric Nanoparticles for Specific Cell Targeting and Drug Delivery","volume":"21","author":"Wang","year":"2020","journal-title":"Biomacromolecules"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"12680","DOI":"10.1039\/C9NR01755K","article-title":"Prion soft amyloid core driven self-assembly of globular proteins into bioactive nanofibrils","volume":"11","author":"Wang","year":"2019","journal-title":"Nanoscale"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1080\/19336896.2020.1785659","article-title":"Prion domains as a driving force for the assembly of functional nanomaterials","volume":"14","author":"Wang","year":"2020","journal-title":"Prion"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1186\/s12934-016-0457-z","article-title":"Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli","volume":"15","author":"Rueda","year":"2016","journal-title":"Microb. Cell Factories"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"13917","DOI":"10.1038\/s41598-018-32213-8","article-title":"A new approach to obtain pure and active proteins from Lactococcus lactis protein aggregates","volume":"8","author":"Fabregas","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1186\/s12934-016-0565-9","article-title":"Functional inclusion bodies produced in the yeast Pichia pastoris","volume":"15","author":"Rueda","year":"2016","journal-title":"Microb. Cell Factories"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1002\/ppp3.10073","article-title":"Therapeutic recombinant protein production in plants: Challenges and opportunities","volume":"2","author":"Burnett","year":"2020","journal-title":"Plants People Planet"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/12\/2\/308\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:19:52Z","timestamp":1760134792000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/12\/2\/308"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,15]]},"references-count":52,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["biom12020308"],"URL":"https:\/\/doi.org\/10.3390\/biom12020308","relation":{},"ISSN":["2218-273X"],"issn-type":[{"type":"electronic","value":"2218-273X"}],"subject":[],"published":{"date-parts":[[2022,2,15]]}}}