{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T10:13:18Z","timestamp":1774260798765,"version":"3.50.1"},"reference-count":51,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2019,2,9]],"date-time":"2019-02-09T00:00:00Z","timestamp":1549670400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["POCI-01-0145-FEDER-030331"],"award-info":[{"award-number":["POCI-01-0145-FEDER-030331"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["POCI-01-0145-FEDER-007440"],"award-info":[{"award-number":["POCI-01-0145-FEDER-007440"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["ProSafe\/0001\/2016"],"award-info":[{"award-number":["ProSafe\/0001\/2016"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Current vaccine research is mostly based on subunit antigens. Despite the better toxicity profile of these antigens they are often poorly immunogenic, so adjuvant association has been explored as a strategy to obtain a potent vaccine formulation. Recently, mast cell activators were recognized as a new class of vaccine adjuvants capable of potentiating mucosal and systemic immune responses. In this study, a co-adjuvanted delivery system was developed and characterized, combining the mast cell activator C48\/80 with chitosan nanoparticles (Chi-C48\/80 NPs), and the results were compared with plain chitosan nanoparticles. The adsorption of model antigens onto the NP surface as well as the biocompatibility of the system was not affected by the incorporation of C48\/80 in the formulation. The stability of the nanoparticles was demonstrated by studying the variation of size and zeta potential at different times, and the ability to be internalized by antigen presenting cells was confirmed by confocal microscopy. Vaccination studies with hepatitis B surface antigen loaded Chi-C48\/80 NPs validated the adjuvanticity of the delivery system, demonstrating for the first time a successful association between a mast cell activator and chitosan nanoparticles as a vaccine adjuvant for hepatitis B virus, applied to a nasal vaccination strategy.<\/jats:p>","DOI":"10.3390\/pharmaceutics11020072","type":"journal-article","created":{"date-parts":[[2019,2,12]],"date-time":"2019-02-12T03:18:20Z","timestamp":1549941500000},"page":"72","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":40,"title":["Chitosan Plus Compound 48\/80: Formulation and Preliminary Evaluation as a Hepatitis B Vaccine Adjuvant"],"prefix":"10.3390","volume":"11","author":[{"given":"Dulce","family":"Bento","sequence":"first","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-0504 Coimbra, Portugal"},{"name":"Faculty of Pharmacy, University of Coimbra, P\u00f3lo das Ci\u00eancias da Sa\u00fade, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4014-9072","authenticated-orcid":false,"given":"Sandra","family":"Jesus","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-0504 Coimbra, Portugal"},{"name":"Faculty of Pharmacy, University of Coimbra, P\u00f3lo das Ci\u00eancias da Sa\u00fade, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3146-269X","authenticated-orcid":false,"given":"Filipa","family":"Lebre","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-0504 Coimbra, Portugal"},{"name":"Faculty of Pharmacy, University of Coimbra, P\u00f3lo das Ci\u00eancias da Sa\u00fade, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9347-0535","authenticated-orcid":false,"given":"Teresa","family":"Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-0504 Coimbra, Portugal"},{"name":"Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2215-5121","authenticated-orcid":false,"given":"Olga","family":"Borges","sequence":"additional","affiliation":[{"name":"CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-0504 Coimbra, Portugal"},{"name":"Faculty of Pharmacy, University of Coimbra, P\u00f3lo das Ci\u00eancias da Sa\u00fade, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2019,2,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1038\/nri3488","article-title":"Applications of nanotechnology for immunology","volume":"13","author":"Smith","year":"2013","journal-title":"Nat. Rev. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1016\/j.drudis.2011.04.006","article-title":"Particulate vaccines: on the quest for optimal delivery and immune response","volume":"16","author":"Rejman","year":"2011","journal-title":"Drug Discov. Today"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4959","DOI":"10.1016\/j.biomaterials.2011.03.049","article-title":"Intracellular degradation and distribution of protein-encapsulated amphiphilic poly(amino acid) nanoparticles","volume":"32","author":"Akagi","year":"2011","journal-title":"Biomaterials"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.1586\/erv.10.89","article-title":"Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses","volume":"9","author":"Oyewumi","year":"2010","journal-title":"Expert Rev. Vaccines"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1111\/j.1365-2567.2005.02268.x","article-title":"Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles","volume":"117","author":"Shen","year":"2006","journal-title":"Immunology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.jconrel.2012.11.015","article-title":"Nanogel vaccines targeting dendritic cells: contributions of the surface decoration and vaccine cargo on cell targeting and activation","volume":"166","author":"Kaeuper","year":"2013","journal-title":"J. Control. Release"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2359","DOI":"10.1016\/j.biomaterials.2012.11.066","article-title":"The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells","volume":"34","author":"Koppolu","year":"2013","journal-title":"Biomaterials"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Gao, Y., Wijewardhana, C., and Mann, J.F.S. (2018). Virus-Like Particle, Liposome, and Polymeric Particle-Based Vaccines against HIV-1. Front. Immunol., 9.","DOI":"10.3389\/fimmu.2018.00345"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Barclay, T., and Petrovsky, N. (2017). Vaccine Adjuvant Nanotechnologies. Micro and Nanotechnology in Vaccine Development, Elsevier Science.","DOI":"10.1016\/B978-0-323-39981-4.00007-5"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1586\/erv.09.47","article-title":"Chitosan-based systems for the delivery of vaccine antigens","volume":"8","author":"Arca","year":"2009","journal-title":"Expert Rev. Vaccines"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.addr.2009.11.009","article-title":"Chitosan-based delivery systems for protein therapeutics and antigens","volume":"62","author":"Amidi","year":"2010","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1016\/j.yrtph.2009.09.015","article-title":"The safety of chitosan as a pharmaceutical excipient","volume":"56","author":"Baldrick","year":"2010","journal-title":"Regul. Toxicol. Pharmacol. RTP"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1586\/erv.10.154","article-title":"Combination adjuvants: the next generation of adjuvants?","volume":"10","author":"Mutwiri","year":"2011","journal-title":"Expert Rev. Vaccines"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1586\/erv.11.21","article-title":"Trends in vaccine adjuvants","volume":"10","author":"Schijns","year":"2011","journal-title":"Expert Rev. Vaccines"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1038\/nri2782","article-title":"Mast cell-orchestrated immunity to pathogens","volume":"10","author":"Abraham","year":"2010","journal-title":"Nat. Rev. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/j.it.2007.03.003","article-title":"Mast cells--key effector cells in immune responses","volume":"28","author":"Metz","year":"2007","journal-title":"Trends Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1038\/nri1460","article-title":"Mast-cell responses to pathogens","volume":"4","author":"Marshall","year":"2004","journal-title":"Nat. Rev. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1038\/nm1757","article-title":"Mast cell activators: A new class of highly effective vaccine adjuvants","volume":"14","author":"McLachlan","year":"2008","journal-title":"Nat. Med."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Staats, H.F., Fielhauer, J.R., Thompson, A.L., Tripp, A.A., Sobel, A.E., Maddaloni, M., Abraham, S.N., and Pascual, D.W. (2011). Mucosal targeting of a BoNT\/A subunit vaccine adjuvanted with a mast cell activator enhances induction of BoNT\/A neutralizing antibodies in rabbits. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0016532"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3544","DOI":"10.1016\/j.vaccine.2009.03.069","article-title":"The mast cell activator compound 48\/80 is safe and effective when used as an adjuvant for intradermal immunization with Bacillus anthracis protective antigen","volume":"27","author":"McGowen","year":"2009","journal-title":"Vaccine"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1002\/jps.22742","article-title":"Stable dry powder formulation for nasal delivery of anthrax vaccine","volume":"101","author":"Wang","year":"2012","journal-title":"J. Pharm. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1038\/nri1439","article-title":"NALT- versus Peyer\u2019s-patch-mediated mucosal immunity","volume":"4","author":"Kiyono","year":"2004","journal-title":"Nat. Rev. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1038\/nri1777","article-title":"Mucosal vaccines: The promise and the challenge","volume":"6","author":"Neutra","year":"2006","journal-title":"Nat. Rev. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.it.2008.09.006","article-title":"New horizons in adjuvants for vaccine development","volume":"30","author":"Reed","year":"2009","journal-title":"Trends Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.colsurfb.2007.04.009","article-title":"Chitosan nanoparticle as protein delivery carrier\u2014Systematic examination of fabrication conditions for efficient loading and release","volume":"59","author":"Gan","year":"2007","journal-title":"Colloids Surf. B Biointerface"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.ijpharm.2017.05.028","article-title":"Association of chitosan and aluminium as a new adjuvant strategy for improved vaccination","volume":"527","author":"Lebre","year":"2017","journal-title":"Int. J. Pharm."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.ijpharm.2016.03.013","article-title":"Immune response elicited by an intranasally delivered HBsAg low-dose adsorbed to poly-epsilon-caprolactone based nanoparticles","volume":"504","author":"Jesus","year":"2016","journal-title":"Int. J. Pharm."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1208\/s12249-013-9950-4","article-title":"Validation of a new 96-well plate spectrophotometric method for the quantification of compound 48\/80 associated with particles","volume":"14","author":"Bento","year":"2013","journal-title":"AAPS PharmSciTech"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1016\/j.jconrel.2006.06.011","article-title":"Uptake studies in rat Peyer\u2019s patches, cytotoxicity and release studies of alginate coated chitosan nanoparticles for mucosal vaccination","volume":"114","author":"Borges","year":"2006","journal-title":"J. Control. Release"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1021\/acs.molpharmaceut.5b00707","article-title":"Intranasal Administration of Novel Chitosan Nanoparticle\/DNA Complexes Induces Antibody Response to Hepatitis B Surface Antigen in Mice","volume":"13","author":"Lebre","year":"2016","journal-title":"Mol. Pharm."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1007\/978-1-4939-3389-1_45","article-title":"Poly-epsilon-caprolactone\/Chitosan and Chitosan Particles: Two Recombinant Antigen Delivery Systems for Intranasal Vaccination","volume":"1404","author":"Jesus","year":"2016","journal-title":"Method Mol. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/j.carbpol.2010.02.026","article-title":"Bioadhesive chitosan nanoparticles: Preparation and characterization","volume":"81","author":"Dudhani","year":"2010","journal-title":"Carbohydr. Polym."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.ijpharm.2005.04.037","article-title":"Preparation of coated nanoparticles for a new mucosal vaccine delivery system","volume":"299","author":"Borges","year":"2005","journal-title":"Int. J. Pharm."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/S0168-3659(99)00285-0","article-title":"Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan","volume":"66","author":"Lim","year":"2000","journal-title":"J. Control. Release"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1016\/j.vaccine.2013.11.069","article-title":"Nanoparticle vaccines","volume":"32","author":"Zhao","year":"2014","journal-title":"Vaccine"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.ijpharm.2005.03.035","article-title":"Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model","volume":"298","author":"Foged","year":"2005","journal-title":"Int. J. Pharm."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1039\/C3AN01684F","article-title":"ATR-FTIR spectroscopy as a tool to probe surface adsorption on nanoparticles at the liquid-solid interface in environmentally and biologically relevant media","volume":"139","author":"Mudunkotuwa","year":"2014","journal-title":"Analyst"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.ijpharm.2013.07.034","article-title":"Chitosan nanoparticles: preparation, size evolution and stability","volume":"455","author":"Rampino","year":"2013","journal-title":"Int. J. Pharm."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.ijpharm.2010.04.003","article-title":"Towards preserving the immunogenicity of protein antigens carried by nanoparticles while avoiding the cold chain","volume":"393","author":"Sloat","year":"2010","journal-title":"Int. J. Pharm."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1688","DOI":"10.1016\/j.addr.2006.09.017","article-title":"Freeze-drying of nanoparticles: formulation, process and storage considerations","volume":"58","author":"Abdelwahed","year":"2006","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Dee, K.C., Puleo, D.A., and Bizios, R. (2003). Protein-Surface Interactions. An Introduction To Tissue-Biomaterial Interactions, John Wiley & Sons, Inc.","DOI":"10.1002\/0471270598"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/S0168-3659(02)00296-1","article-title":"Stimulation of spleen cells in vitro by nanospheric particles containing antigen","volume":"86","author":"Eyles","year":"2003","journal-title":"J. Control. Release"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.ejps.2007.02.008","article-title":"Chitosan nanoparticles are compatible with respiratory epithelial cells in vitro","volume":"31","author":"Grenha","year":"2007","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.addr.2009.09.004","article-title":"Biodegradation, biodistribution and toxicity of chitosan","volume":"62","author":"Kean","year":"2010","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1038\/nrd1176","article-title":"Recent advances in the discovery and delivery of vaccine adjuvants","volume":"2","author":"Valiante","year":"2003","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2440","DOI":"10.1021\/bm101482r","article-title":"Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles","volume":"12","author":"Yue","year":"2011","journal-title":"Biomacromolecules"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/j.ijpharm.2018.09.044","article-title":"Mechanistic study of the adjuvant effect of chitosan-aluminum nanoparticles","volume":"552","author":"Lebre","year":"2018","journal-title":"Int. J. Pharm."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2335","DOI":"10.2217\/nnm-2017-0138","article-title":"Poly--caprolactone\/chitosan nanoparticles provide strong adjuvant effect for hepatitis B antigen","volume":"12","author":"Jesus","year":"2017","journal-title":"Nanomedicine"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1021\/acs.molpharmaceut.7b00730","article-title":"Adjuvant Activity of Poly-epsilon-caprolactone\/Chitosan Nanoparticles Characterized by Mast Cell Activation and IFN-gamma and IL-17 Production","volume":"15","author":"Jesus","year":"2018","journal-title":"Mol. Pharm."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Schubert, N., Lisenko, K., Auerbach, C., Weitzmann, A., Ghouse, S.M., Muhandes, L., Haase, C., H\u00e4ring, T., Schulze, L., and Voehringer, D. (2018). Unimpaired Responses to Vaccination With Protein Antigen Plus Adjuvant in Mice With Kit-Independent Mast Cell Deficiency. Front. Immunol., 9.","DOI":"10.3389\/fimmu.2018.01870"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/j.ejpb.2015.03.024","article-title":"Development of a novel adjuvanted nasal vaccine: C48\/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity","volume":"93","author":"Bento","year":"2015","journal-title":"Eur. J. Pharm. Biopharm."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/11\/2\/72\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T12:30:50Z","timestamp":1760185850000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/11\/2\/72"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,2,9]]},"references-count":51,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2019,2]]}},"alternative-id":["pharmaceutics11020072"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics11020072","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,2,9]]}}}