{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T18:18:48Z","timestamp":1772821128341,"version":"3.50.1"},"reference-count":60,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,3,14]],"date-time":"2023-03-14T00:00:00Z","timestamp":1678752000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Marine Drugs"],"abstract":"<jats:p>The design of nanoparticle formulations composed of biopolymers, that govern the physicochemical properties of orally delivered insulin, relies on improving insulin stability and absorption through the intestinal mucosa while protecting it from harsh conditions in the gastrointestinal (GI) tract. Chitosan\/polyethylene glycol (PEG) and albumin coating of alginate\/dextran sulfate hydrogel cores are presented as a multilayer complex protecting insulin within the nanoparticle. This study aims to optimize a nanoparticle formulation by assessing the relationship between design parameters and experimental data using response surface methodology through a 3-factor 3-level optimization Box\u2013Behnken design. While the selected independent variables were the concentrations of PEG, chitosan and albumin, the dependent variables were particle size, polydispersity index (PDI), zeta potential, and insulin release. Experimental results showed a nanoparticle size ranging from 313 to 585 nm, with PDI from 0.17 to 0.39 and zeta potential ranging from \u221229 to \u221244 mV. Insulin bioactivity was maintained in simulated GI media with over 45% cumulative release after 180 min in a simulated intestinal medium. Based on the experimental responses and according to the criteria of desirability on the experimental region\u2019s constraints, solutions of 0.03% PEG, 0.047% chitosan and 1.20% albumin provide an optimum nanoparticle formulation for insulin oral delivery.<\/jats:p>","DOI":"10.3390\/md21030179","type":"journal-article","created":{"date-parts":[[2023,3,15]],"date-time":"2023-03-15T04:39:45Z","timestamp":1678855185000},"page":"179","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Chitosan\/Albumin Coating Factorial Optimization of Alginate\/Dextran Sulfate Cores for Oral Delivery of Insulin"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6435-886X","authenticated-orcid":false,"given":"Bruno","family":"Pessoa","sequence":"first","affiliation":[{"name":"Faculty of Pharmacy, University of Coimbra, 3000-048 Coimbra, Portugal"}]},{"given":"Mar","family":"Collado-Gonzalez","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, University of Coimbra, 3000-048 Coimbra, Portugal"},{"name":"Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6766-9321","authenticated-orcid":false,"given":"Giuseppina","family":"Sandri","sequence":"additional","affiliation":[{"name":"Department of Drug Sciences, University of Pavia, Viale Taramelli, 12, 27100 Pavia, Italy"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1399-8944","authenticated-orcid":false,"given":"Ant\u00f3nio","family":"Ribeiro","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, University of Coimbra, 3000-048 Coimbra, Portugal"},{"name":"i<sub>3<\/sub>S, IBMC, Rua Alfredo Allen, 4200-135 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1007\/s00125-021-05396-5","article-title":"Transforming Type 1 Diabetes: The next Wave of Innovation","volume":"64","author":"Drucker","year":"2021","journal-title":"Diabetologia"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"102615","DOI":"10.1016\/j.dsx.2022.102615","article-title":"Once-Weekly Basal Insulin Icodec: Looking ONWARDS from Pharmacology to Clinical Trials","volume":"16","author":"Singh","year":"2022","journal-title":"Diabetes Metab. Syndr. Clin. Res. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/S2213-8587(18)30372-3","article-title":"Efficacy and Safety of Oral Basal Insulin versus Subcutaneous Insulin Glargine in Type 2 Diabetes: A Randomised, Double-Blind, Phase 2 Trial","volume":"7","author":"Halberg","year":"2019","journal-title":"Lancet Diabetes Endocrinol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1038\/s41573-019-0053-0","article-title":"Advances in Oral Peptide Therapeutics","volume":"19","author":"Drucker","year":"2020","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_5","unstructured":"(2023, February 01). Oramed Announces Top-Line Results from Phase 3 Trial of ORMD-0801 for the Treatment of Type 2 Diabetes\u2014Oramed Pharmaceuticals. Available online: https:\/\/oramed.com\/oramed-announces-top-line-results-from-phase-3-trial-of-ormd-0801-for-the-treatment-of-type-2-diabetes\/."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"973","DOI":"10.4155\/TDE.15.47","article-title":"Why Most Oral Insulin Formulations Do Not Reach Clinical Trials","volume":"6","author":"Lopes","year":"2015","journal-title":"Ther. Deliv."},{"key":"ref_7","first-page":"109","article-title":"Nanoparticles for Oral Delivery of Insulin","volume":"Volume 4","author":"Coelho","year":"2013","journal-title":"Drug Delivery Systems: Advanced Technologies Potentially Applicable in Personalised Treatment"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"589","DOI":"10.2174\/1389201015666140915153104","article-title":"Editorial Thematic Issue: Nanotechnology as a Strategy to Improve Treatment of Diabetes","volume":"15","author":"Ribeiro","year":"2014","journal-title":"Curr. Pharm. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1186\/s12951-018-0392-8","article-title":"Nano Based Drug Delivery Systems: Recent Developments and Future Prospects","volume":"16","author":"Patra","year":"2018","journal-title":"J. Nanobiotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.nano.2006.04.009","article-title":"Nanoencapsulation II. Biomedical Applications and Current Status of Peptide and Protein Nanoparticulate Delivery Systems","volume":"2","author":"Neufeld","year":"2006","journal-title":"Nanomedicine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"672","DOI":"10.2174\/1567201816666190620110748","article-title":"Chitosan-Coated Alginate Nanoparticles Enhanced Absorption Profile of Insulin via Oral Administration","volume":"16","author":"Jaafar","year":"2019","journal-title":"Curr. Drug Deliv."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1683","DOI":"10.1016\/j.ijbiomac.2020.07.276","article-title":"Sterculia Striata Gum as a Potential Oral Delivery System for Protein Drugs","volume":"164","author":"Freitas","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Collado-Gonz\u00e1lez, M., Ferreri, M.C., Freitas, A.R., Santos, A.C., Ferreira, N.R., Carissimi, G., Sequeira, J.A.D., D\u00edaz Ba\u00f1os, F.G., Villora, G., and Veiga, F. (2020). Complex Polysaccharide-Based Nanocomposites for Oral Insulin Delivery. Mar. Drugs, 18.","DOI":"10.3390\/md18010055"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Vodyashkin, A.A., Kezimana, P., Vetcher, A.A., and Stanishevskiy, Y.M. (2022). Biopolymeric Nanoparticles\u2014Multifunctional Materials of the Future. Polymers, 14.","DOI":"10.3390\/polym14112287"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2833","DOI":"10.1166\/jnn.2007.609","article-title":"Insulin-Loaded Nanoparticles Are Prepared by Alginate Ionotropic Pre-Gelation Followed by Chitosan Polyelectrolyte Complexation","volume":"7","author":"Sarmento","year":"2007","journal-title":"J. Nanosci. Nanotechnol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1080\/17425247.2016.1214564","article-title":"Preparation Methods and Applications behind Alginate-Based Particles","volume":"14","author":"Lopes","year":"2017","journal-title":"Expert. Opin. Drug Deliv."},{"key":"ref_17","unstructured":"Benediktsd\u00f3ttir, B.E. (2020). Encyclopedia of Marine Biotechnology, Wiley."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5865","DOI":"10.2147\/IJN.S86313","article-title":"Probing Insulin Bioactivity in Oral Nanoparticles Produced by Ultrasonication-Assisted Emulsification\/Internal Gelation","volume":"10","author":"Ribeiro","year":"2015","journal-title":"Int. J. Nanomed."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"20155","DOI":"10.1039\/C5RA26224K","article-title":"Impact of the in Vitro Gastrointestinal Passage of Biopolymer-Based Nanoparticles on Insulin Absorption","volume":"6","author":"Lopes","year":"2016","journal-title":"RSC Adv."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.jconrel.2016.04.012","article-title":"Dual Chitosan\/Albumin-Coated Alginate\/Dextran Sulfate Nanoparticles for Enhanced Oral Delivery of Insulin","volume":"232","author":"Lopes","year":"2016","journal-title":"J. Control Rel."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1007\/s10989-004-2433-4","article-title":"Chitosan-Dextran Sulfate Nanoparticles for Delivery of an Anti-Angiogenesis Peptide","volume":"10","author":"Chen","year":"2003","journal-title":"Int. J. Pept. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"145","DOI":"10.17113\/ftb.60.02.22.6981","article-title":"Polyethylene Glycol-Stabilized Zein Nanoparticles Containing Gallic Acid","volume":"60","author":"Wiggers","year":"2022","journal-title":"Food Technol. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1080\/17435390802398309","article-title":"Toxicological Assessment of Orally Delivered Nanoparticulate Insulin","volume":"2","author":"Reis","year":"2008","journal-title":"Nanotoxicology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1862","DOI":"10.1021\/acs.molpharmaceut.1c00046","article-title":"The Uniqueness of Albumin as a Carrier in Nanodrug Delivery","volume":"18","author":"Spada","year":"2021","journal-title":"Mol. Pharm."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4695","DOI":"10.1038\/s41598-018-23064-4","article-title":"Parameters Influencing the Size of Chitosan-TPP Nano- and Microparticles","volume":"8","author":"Sreekumar","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5419","DOI":"10.1002\/jsfa.11190","article-title":"Formulation and Characterization of Zein\/Chitosan Complex Particles Stabilized Pickering Emulsion with the Encapsulation and Delivery of Vitamin D3","volume":"101","author":"Shah","year":"2021","journal-title":"J. Sci. Food Agric."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/j.colsurfb.2011.02.020","article-title":"Preparation and Characterization of Zein\/Chitosan Complex for Encapsulation of \u03b1-Tocopherol, and Its in Vitro Controlled Release Study","volume":"85","author":"Luo","year":"2011","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5246","DOI":"10.1038\/s41467-021-25584-6","article-title":"The Need for Robust Characterization of Nanomaterials for Nanomedicine Applications","volume":"12","author":"Mahmoudi","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.jconrel.2015.05.005","article-title":"Development of a Gene-Activated Scaffold Platform for Tissue Engineering Applications Using Chitosan-PDNA Nanoparticles on Collagen-Based Scaffolds","volume":"210","author":"Raftery","year":"2015","journal-title":"J. Control. Rel."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1016\/j.carbpol.2008.11.025","article-title":"Macromolecular Conformation of Chitosan in Dilute Solution: A New Global Hydrodynamic Approach","volume":"76","author":"Morris","year":"2009","journal-title":"Carbohydr. Polym."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.jcis.2008.12.002","article-title":"Functionalized Gold Nanoparticles: Synthesis, Structure and Colloid Stability","volume":"331","author":"Zhou","year":"2009","journal-title":"J. Colloid Interface Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.1016\/j.ijbiomac.2020.04.118","article-title":"PEGylation and Folate Conjugation Effects on the Stability of Chitosan-Tripolyphosphate Nanoparticles","volume":"158","author":"Gartner","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Amin, M.K., and Boateng, J.S. (2022). Enhancing Stability and Mucoadhesive Properties of Chitosan Nanoparticles by Surface Modification with Sodium Alginate and Polyethylene Glycol for Potential Oral Mucosa Vaccine Delivery. Mar. Drugs, 20.","DOI":"10.3390\/md20030156"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"20200190","DOI":"10.1002\/VIW.20200190","article-title":"Consideration for the Scale-up Manufacture of Nanotherapeutics\u2014A Critical Step for Technology Transfer","volume":"2","author":"Liu","year":"2021","journal-title":"VIEW"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1021\/acs.molpharmaceut.1c00699","article-title":"Design of Experiment Approach to Modeling the Effects of Formulation and Drug Loading on the Structure and Properties of Therapeutic Nanogels","volume":"19","author":"Ho","year":"2022","journal-title":"Mol. Pharm."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1016\/j.ejpb.2021.05.011","article-title":"Design of Experiments (DoE) to Develop and to Optimize Nanoparticles as Drug Delivery Systems","volume":"165","author":"Viegas","year":"2021","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"676","DOI":"10.2174\/1567201813666160801125235","article-title":"Loteprednol Etabonate Nanoparticles: Optimization via Box-Behnken Design Response Surface Methodology and Physicochemical Characterization","volume":"14","author":"Sah","year":"2017","journal-title":"Curr. Drug Deliv."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Beg, S., and Akhter, S. (2021). Design of Experiments for Pharmaceutical Product Development, Springer.","DOI":"10.1007\/978-981-33-4717-5"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.ejpb.2009.06.002","article-title":"Design for Optimization of Nanoparticles Integrating Biomaterials for Orally Dosed Insulin","volume":"73","author":"Woitiski","year":"2009","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"121829","DOI":"10.1016\/j.ijpharm.2022.121829","article-title":"IgG Functionalized Polymeric Nanoparticles for Oral Insulin Administration","volume":"622","author":"Onzi","year":"2022","journal-title":"Int. J. Pharm."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"118720","DOI":"10.1016\/j.ijpharm.2019.118720","article-title":"Bio-Nanotechnological Advancement of Orally Administered Insulin Nanoparticles: Comprehensive Review of Experimental Design for Physicochemical Characterization","volume":"572","author":"Wong","year":"2019","journal-title":"Int. J. Pharm."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.1016\/0142-9612(96)84680-1","article-title":"Alginate Polycation Microcapsules","volume":"17","author":"Thu","year":"1996","journal-title":"Biomaterials"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"5290","DOI":"10.1002\/jps.21347","article-title":"Nanoparticulate Biopolymers Deliver Insulin Orally Eliciting Pharmacological Response","volume":"97","author":"Reis","year":"2008","journal-title":"J. Pharm. Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2475","DOI":"10.1016\/j.actbio.2009.03.007","article-title":"Colloidal Carrier Integrating Biomaterials for Oral Insulin Delivery: Influence of Component Formulation on Physicochemical and Biological Parameters","volume":"5","author":"Woitiski","year":"2009","journal-title":"Acta Biomater."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3054","DOI":"10.1021\/bm0703923","article-title":"Oral Bioavailability of Insulin Contained in Polysaccharide Nanoparticles","volume":"8","author":"Sarmento","year":"2007","journal-title":"Biomacromolecules"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.carbpol.2017.08.068","article-title":"Insight into the Interaction between Chitosan and Bovine Serum Albumin","volume":"176","author":"Li","year":"2017","journal-title":"Carbohydr. Polym."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"107137","DOI":"10.1016\/j.foodhyd.2021.107137","article-title":"PH Influence on the Mechanisms of Interaction between Chitosan and Ovalbumin: A Multi-Spectroscopic Approach","volume":"123","author":"Balbino","year":"2022","journal-title":"Food Hydrocoll."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1002\/jps.2600821102","article-title":"Submicron Emulsions as Colloidal Drug Carriers for Intravenous Administration: Comprehensive Physicochemical Characterization","volume":"82","author":"Benita","year":"1993","journal-title":"J. Pharm. Sci."},{"key":"ref_49","first-page":"255","article-title":"Effect of Zeta Potential on the Properties of Nano-Drug Delivery Systems\u2014A Review (Part 1)","volume":"12","author":"Honary","year":"2013","journal-title":"Trop. J. Pharm. Res."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"16495","DOI":"10.1021\/acs.jpcc.9b00913","article-title":"What Does Nanoparticle Stability Mean?","volume":"123","author":"Phan","year":"2019","journal-title":"J. Phys. Chem. C"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"640","DOI":"10.1016\/j.ijbiomac.2014.08.040","article-title":"PH-Sensitive Chitosan\/Alginate Core-Shell Nanoparticles for Efficient and Safe Oral Insulin Delivery","volume":"72","author":"Mukhopadhyay","year":"2015","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1016\/j.msec.2016.08.083","article-title":"Preparation of Chitosan-Based Multifunctional Nanocarriers Overcoming Multiple Barriers for Oral Delivery of Insulin","volume":"70","author":"Li","year":"2017","journal-title":"Mater. Sci. Eng. C"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1017\/S003358351000003X","article-title":"Protein Characterisation by Synchrotron Radiation Circular Dichroism Spectroscopy","volume":"42","author":"Wallace","year":"2009","journal-title":"Q. Rev. Biophys."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"4259","DOI":"10.1021\/acs.langmuir.6b00919","article-title":"Evaluation of High-Yield Purification Methods on Monodisperse PEG-Grafted Iron Oxide Nanoparticles","volume":"32","author":"Lassenberger","year":"2016","journal-title":"Langmuir"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1021\/bm025689+","article-title":"Improvement in Ductility of Chitosan through Blending and Copolymerization with PEG: FTIR Investigation of Molecular Interactions","volume":"4","author":"Kolhe","year":"2003","journal-title":"Biomacromolecules"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1089\/adt.2021.084","article-title":"Formulation of Lipid-Based Nanocarriers of Lacidipine for Improvement of Oral Delivery: Box-Behnken Design Optimization, In Vitro, Ex Vivo, and Preclinical Assessment","volume":"20","author":"Kataria","year":"2022","journal-title":"Assay. Drug Dev. Technol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1002\/jps.2600820909","article-title":"Development of a New Drug Carrier Made from Alginate","volume":"82","author":"Rajaonarivony","year":"1993","journal-title":"J. Pharm. Sci."},{"key":"ref_58","unstructured":"The United States Pharmacopeia 36 (USP- 36) (1998). National Formulary 31 (NF-31), United States Pharmacopeial Convention."},{"key":"ref_59","first-page":"513","article-title":"Chitosan-Sodium Alginate Nanoparticles as Submicroscopic Reservoirs for Ocular Delivery: Formulation, Optimisation and in Vitro Characterisation","volume":"68","author":"Motwani","year":"2008","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_60","unstructured":"Myers, R.H., Montgomery, D.C., and Anderson-Cook, C.M. (2016). Response Surface Methodology: Process and Product Optimization Using Designed Experiments, Wiley."}],"container-title":["Marine Drugs"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1660-3397\/21\/3\/179\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:55:01Z","timestamp":1760122501000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1660-3397\/21\/3\/179"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,14]]},"references-count":60,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2023,3]]}},"alternative-id":["md21030179"],"URL":"https:\/\/doi.org\/10.3390\/md21030179","relation":{},"ISSN":["1660-3397"],"issn-type":[{"value":"1660-3397","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,3,14]]}}}