{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:49:35Z","timestamp":1760237375275,"version":"build-2065373602"},"reference-count":63,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2020,4,5]],"date-time":"2020-04-05T00:00:00Z","timestamp":1586044800000},"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>Nanoscale drug delivery systems exhibit a broad range of applications and promising treatment possibilities for various medical conditions. Nanomedicine is of great interest, particularly for rare diseases still lacking a curative treatment such as cystic fibrosis (CF). CF is defined by a lack of Cl\u2212 secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) and an increased Na+ absorption mediated by the epithelial sodium channel (ENaC). The imbalanced ion and water transport leads to pathological changes in many organs, particularly in the lung. We developed a non-viral delivery system based on the natural aminopolysaccharide chitosan (CS) for the transport of antisense oligonucleotides (ASO) against ENaC to specifically address Na+ hyperabsorption. CS\u2013ASO electrostatic self-assembled nanocomplexes were formed at varying positive\/negative (P\/N) charge ratios and characterized for their physicochemical properties. Most promising nanocomplexes (P\/N 90) displayed an average size of ~150 nm and a zeta potential of ~+30 mV. Successful uptake of the nanocomplexes by the human airway epithelial cell line NCI-H441 was confirmed by fluorescence microscopy. Functional Ussing chamber measurements of transfected NCI-H441 cells showed significantly decreased Na+ currents, indicating successful downregulation of ENaC. The results obtained confirm the promising characteristics of CS as a non-viral and non-toxic delivery system and demonstrate the encouraging possibility to target ENaC with ASOs to treat abnormal ion transport in CF.<\/jats:p>","DOI":"10.3390\/biom10040553","type":"journal-article","created":{"date-parts":[[2020,4,7]],"date-time":"2020-04-07T03:58:39Z","timestamp":1586231919000},"page":"553","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells"],"prefix":"10.3390","volume":"10","author":[{"given":"A. Katharina","family":"Kolonko","sequence":"first","affiliation":[{"name":"Institute of Animal Physiology, University of Muenster, Schlossplatz 8, 48143 Muenster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nadine","family":"Bangel-Ruland","sequence":"additional","affiliation":[{"name":"Institute of Animal Physiology, University of Muenster, Schlossplatz 8, 48143 Muenster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7949-5429","authenticated-orcid":false,"given":"Francisco M.","family":"Goycoolea","sequence":"additional","affiliation":[{"name":"School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wolf-Michael","family":"Weber","sequence":"additional","affiliation":[{"name":"Institute of Animal Physiology, University of Muenster, Schlossplatz 8, 48143 Muenster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,4,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1038\/nrg3763","article-title":"Non-viral vectors for gene-based therapy","volume":"15","author":"Yin","year":"2014","journal-title":"Nat. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"i859","DOI":"10.1136\/bmj.i859","article-title":"New and emerging targeted therapies for cystic fibrosis","volume":"352","author":"Quon","year":"2016","journal-title":"BMJ"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1066","DOI":"10.1126\/science.2475911","article-title":"Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA","volume":"245","author":"Riordan","year":"1989","journal-title":"Science"},{"key":"ref_4","first-page":"564","article-title":"Cystic fibrosis","volume":"114","author":"Naehrig","year":"2017","journal-title":"Dtsch. Arztebl. Int."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1152\/physrev.1999.79.1.S145","article-title":"CFTR is a conductance regulator as well as a chloride channel","volume":"79","author":"Schwiebert","year":"1999","journal-title":"Physiol. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.jcf.2007.07.012","article-title":"Upregulated expression of ENaC in human CF nasal epithelium","volume":"7","author":"Bangel","year":"2008","journal-title":"J. Cyst. Fibros."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1159\/000016298","article-title":"Minor Role of Cl\u2212 secretion in non-cystic fibrosis and cystic fibrosis human nasal epithelium","volume":"9","author":"Weber","year":"1999","journal-title":"Cell. Physiol. Biochem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1359","DOI":"10.1016\/S0169-409X(02)00144-8","article-title":"An overview of the pathogenesis of cystic fibrosis lung disease","volume":"54","author":"Boucher","year":"2002","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/nrg3849","article-title":"Cystic fibrosis genetics: From molecular understanding to clinical application","volume":"16","author":"Cutting","year":"2015","journal-title":"Nat. Rev. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1002\/jgm.1363","article-title":"Specific inhibition of epithelial Na+ channels by antisense oligonucleotides for the treatment of Na+ hyperabsorption in cystic fibrosis","volume":"11","author":"Sobczak","year":"2009","journal-title":"J. Gene Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/nrneurol.2017.148","article-title":"Antisense oligonucleotides: The next frontier for treatment of neurological disorders","volume":"14","author":"Rinaldi","year":"2018","journal-title":"Nat. Rev. Neurol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.jcf.2019.05.014","article-title":"Antisense oligonucleotide eluforsen is safe and improves respiratory symptoms in F508DEL cystic fibrosis","volume":"19","author":"Drevinek","year":"2020","journal-title":"J. Cyst. Fibros."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1133","DOI":"10.3390\/md13031133","article-title":"Chitin and chitosan preparation from marine sources. Structure, properties and applications","volume":"13","author":"Younes","year":"2015","journal-title":"Mar. Drugs"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/S0169-409X(01)00198-3","article-title":"Chitosans for gene delivery","volume":"52","author":"Borchard","year":"2001","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/S1773-2247(10)50004-2","article-title":"The potential of chitosan for pulmonary drug delivery","volume":"20","author":"Grenha","year":"2010","journal-title":"J. Drug Deliv. Sci. Technol."},{"key":"ref_16","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_17","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1517\/17425247.2.5.843","article-title":"The potential of chitosan for the oral administration of peptides","volume":"2","author":"Prego","year":"2005","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1569","DOI":"10.1016\/j.addr.2005.07.002","article-title":"Thiomers: A new generation of mucoadhesive polymers","volume":"57","year":"2005","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1016\/j.colsurfb.2016.02.031","article-title":"Anti-inflammatory activity of chitosan nanoparticles carrying NF-\u03baB\/p65 antisense oligonucleotide in RAW264.7 macropghage stimulated by lipopolysaccharide","volume":"142","author":"Ma","year":"2016","journal-title":"Colloids Surfaces B Biointerfaces"},{"key":"ref_20","first-page":"17","article-title":"Mannosylated chitosan nanoparticles for delivery of antisense oligonucleotides for macrophage targeting","volume":"2014","author":"Asthana","year":"2014","journal-title":"Biomed Res. Int."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3695","DOI":"10.1166\/jnn.2007.041","article-title":"Chitosan-TPP nanoparticle as a release system of antisense oligonucleotide in the oral environment","volume":"7","author":"Dung","year":"2007","journal-title":"J. Nanosci. Nanotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/j.nano.2007.03.006","article-title":"Chitosan-coated PLGA nanoparticles for DNA\/RNA delivery: Effect of the formulation parameters on complexation and transfection of antisense oligonucleotides","volume":"3","author":"Nafee","year":"2007","journal-title":"Nanomedicine"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ijpharm.2016.01.083","article-title":"Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line","volume":"502","author":"Weber","year":"2016","journal-title":"Int. J. Pharm."},{"key":"ref_24","first-page":"1016","article-title":"Functional restoring of defect CFTR by transfection of CFTR- mRNA using chitosan","volume":"3","author":"Kolonko","year":"2016","journal-title":"JSM Genet. Genomics"},{"key":"ref_25","first-page":"228","article-title":"Specific effects of antisense oligonucleotides on ENaC expressed in Xenopus laevis oocytes","volume":"443","author":"Segal","year":"2002","journal-title":"Pflugers Arch."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2295","DOI":"10.1002\/macp.200800290","article-title":"Electrostatic self-assembly as route to supramolecular structures","volume":"209","year":"2008","journal-title":"Macromol. Chem. Phys."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1007\/s00396-012-2604-3","article-title":"Effects of ionic strength on the size and compactness of chitosan nanoparticles","volume":"290","author":"Jonassen","year":"2012","journal-title":"Colloid Polym. Sci."},{"key":"ref_28","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_29","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.colsurfb.2017.05.055","article-title":"Preparation of chitosan\/tripolyphosphate nanoparticles with highly tunable size and low polydispersity","volume":"157","author":"Sawtarie","year":"2017","journal-title":"Colloids Surfaces B Biointerfaces"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"13567","DOI":"10.1038\/srep13567","article-title":"Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells","volume":"5","author":"Aaldering","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"10392","DOI":"10.1021\/la201194a","article-title":"Monovalent salt enhances colloidal stability during the formation of chitosan\/tripolyphosphate microgels","volume":"27","author":"Huang","year":"2011","journal-title":"Langmuir"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.ijpharm.2010.09.041","article-title":"Optimization of formulation and process variable of nanosuspension: An industrial perspective","volume":"402","author":"Singare","year":"2010","journal-title":"Int. J. Pharm."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1023\/A:1011037807261","article-title":"Water-soluble and low molecular weight chitosan-based plasmid DNA delivery","volume":"18","author":"Lee","year":"2001","journal-title":"Pharm. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/s10856-018-6160-3","article-title":"Chitosan\/hyaluronic acid\/plasmid-DNA nanoparticles encoding interleukin-1 receptor antagonist attenuate inflammation in synoviocytes induced by interleukin-1 beta","volume":"29","author":"Deng","year":"2018","journal-title":"J. Mater. Sci. Mater. Med."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.addr.2008.09.011","article-title":"Extracellular barriers in respiratory gene therapy","volume":"61","author":"Sanders","year":"2009","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"708","DOI":"10.1016\/j.pupt.2006.08.008","article-title":"Actin limits enhancement of nanoparticle diffusion through cystic fibrosis sputum by mucolytics","volume":"20","author":"Smith","year":"2007","journal-title":"Pulm. Pharmacol. Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1905","DOI":"10.1164\/ajrccm.162.5.9909009","article-title":"Cystic fibrosis sputum","volume":"162","author":"Sanders","year":"2000","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ajps.2013.07.001","article-title":"The endocytosis and intracellular fate of nanomedicines: Implication for rational design","volume":"8","author":"Kou","year":"2013","journal-title":"Asian J. Pharm. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.jconrel.2005.05.004","article-title":"Transfection efficiency of chitosan vectors: Effect of polymer molecular weight and degree of deacetylation","volume":"106","author":"Huang","year":"2005","journal-title":"J. Control. Release"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"398","DOI":"10.1080\/09205063.2012.690282","article-title":"Modeling the proton sponge hypothesis: Examining proton sponge effectiveness for enhancing intracellular gene delivery through multiscale modeling","volume":"24","author":"Freeman","year":"2013","journal-title":"J. Biomater. Sci. Polym. Ed."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Funkhouser, J.D., and Aronson, N.N. (2007). Chitinase family GH18: Evolutionary insights from the genomic history of a diverse protein family. BMC Evol. Biol., 7.","DOI":"10.1186\/1471-2148-7-96"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"6770","DOI":"10.1074\/jbc.M009886200","article-title":"Identification of a novel acidic mammalian chitinase distinct from chitotriosidase","volume":"276","author":"Boot","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.jbiotec.2015.04.024","article-title":"Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection","volume":"208","author":"Liang","year":"2015","journal-title":"J. Biotechnol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"44134","DOI":"10.1038\/srep44134","article-title":"Size matters for in vitro gene delivery: Investigating the relationships among complexation protocol, transfection medium, size and sedimentation","volume":"7","author":"Pezzoli","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.omtm.2018.12.005","article-title":"Utilization of HEPES for enhancing protein transfection into mammalian cells","volume":"13","author":"Chen","year":"2019","journal-title":"Mol. Ther. - Methods Clin. Dev."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Riedl, S., Kaiser, P., Raup, A., Synatschke, C., J\u00e9r\u00f4me, V., and Freitag, R. (2018). Non-viral transfection of human T lymphocytes. Processes, 6.","DOI":"10.3390\/pr6100188"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Fern\u00e1ndez Fern\u00e1ndez, E., Santos-Carballal, B., de Santi, C., Ramsey, J., MacLoughlin, R., Cryan, S.-A., and Greene, C. (2018). Biopolymer-based nanoparticles for cystic fibrosis lung gene therapy studies. Materials, 11.","DOI":"10.3390\/ma11010122"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1007\/BF02616113","article-title":"Osmolality of mammalian blood and of media for culture of mammalian cells","volume":"6","author":"Waymouth","year":"1970","journal-title":"In Vitro"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1002\/cyto.a.23082","article-title":"Quantitative phase imaging for cell culture quality control","volume":"91","author":"Kastl","year":"2017","journal-title":"Cytom. Part A"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1165\/2009-0147OC","article-title":"AMPK agonists ameliorate sodium and fluid transport and inflammation in cystic fibrosis airway epithelial cells","volume":"42","author":"Myerburg","year":"2010","journal-title":"Am. J. Respir. Cell Mol. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"L88","DOI":"10.1152\/ajplung.00142.2010","article-title":"Regulation of endogenous ENaC functional expression by CFTR and \u0394F508-CFTR in airway epithelial cells","volume":"300","author":"Rubenstein","year":"2011","journal-title":"Am. J. Physiol. Cell. Mol. Physiol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.ejpb.2017.03.020","article-title":"Modelling the bronchial barrier in pulmonary drug delivery: A human bronchial epithelial cell line supplemented with human tracheal mucus","volume":"118","author":"Murgia","year":"2017","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"12431","DOI":"10.1074\/jbc.274.18.12431","article-title":"Glucocorticoid induction of epithelial sodium channel expression in lung and renal epithelia occurs via trans-activation of a hormone response element in the 5\u2032-flanking region of the human epithelial sodium channel alpha subunit gene","volume":"274","author":"Sayegh","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"L844","DOI":"10.1152\/ajplung.00355.2002","article-title":"Development of cystic fibrosis and noncystic fibrosis airway cell lines","volume":"284","author":"Zabner","year":"2003","journal-title":"Am. J. Physiol. Cell. Mol. Physiol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1021\/cr00102a001","article-title":"Antisense oligonucleotides: A new therapeutic principle","volume":"90","author":"Uhlmann","year":"1990","journal-title":"Chem. Rev."},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Wang, T., Larcher, L., Ma, L., and Veedu, R. (2018). Systematic screening of commonly used commercial transfection reagents towards efficient transfection of single-stranded oligonucleotides. Molecules, 23.","DOI":"10.3390\/molecules23102564"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1038\/gt.2017.13","article-title":"Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent","volume":"24","author":"Chernousova","year":"2017","journal-title":"Gene Ther."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1208\/pt020420","article-title":"In vivo and in vitro characterization of novel microparticulates based on hyaluronan and chitosan hydroglutamate","volume":"2","author":"Lim","year":"2001","journal-title":"AAPS PharmSciTech"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1016\/j.jconrel.2005.10.001","article-title":"Enhancement of bronchial octreotide absorption by chitosan and N-trimethyl chitosan shows linear in vitro\/in vivo correlation","volume":"110","author":"Florea","year":"2006","journal-title":"J. Control. Release"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"L1046","DOI":"10.1152\/ajplung.1999.276.6.L1046","article-title":"Antisense oligonucleotides against the \u03b1-subunit of ENaC decrease lung epithelial cation-channel activity","volume":"276","author":"Jain","year":"1999","journal-title":"Am. J. Physiol. Cell. Mol. Physiol."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/1465-9921-7-26","article-title":"Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo","volume":"7","author":"Griesenbach","year":"2006","journal-title":"Respir. Res."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/j.jcf.2017.05.003","article-title":"Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice","volume":"16","author":"Crosby","year":"2017","journal-title":"J. Cyst. Fibros."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"1475","DOI":"10.3389\/fphar.2018.01475","article-title":"Animal models in the pathophysiology of cystic fibrosis","volume":"9","author":"Semaniakou","year":"2018","journal-title":"Front. Pharmacol."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/4\/553\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:15:44Z","timestamp":1760174144000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/10\/4\/553"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,4,5]]},"references-count":63,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2020,4]]}},"alternative-id":["biom10040553"],"URL":"https:\/\/doi.org\/10.3390\/biom10040553","relation":{},"ISSN":["2218-273X"],"issn-type":[{"type":"electronic","value":"2218-273X"}],"subject":[],"published":{"date-parts":[[2020,4,5]]}}}