{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T22:48:36Z","timestamp":1782514116816,"version":"3.54.5"},"reference-count":26,"publisher":"Springer Science and Business Media LLC","issue":"7","license":[{"start":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T00:00:00Z","timestamp":1776211200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T00:00:00Z","timestamp":1776211200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100009088","name":"University of Veterinary Medicine Vienna","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100009088","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Arch Toxicol"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>RNA therapeutics hold strong potential for treating genetic disease, yet progress is often limited by delivery, stability, and safety concerns. Here, we profile respiratory safety and cellular uptake of an inhaled siRNA targeting the cardiac RyR2 gene, which is responsible for catecholaminergic polymorphic ventricular tachycardia (CPVT), delivered via inhalation with calcium phosphate nanoparticles (CaP NPs). Barrier integrity, cytotoxicity, siRNA uptake, and immune activation, were assessed using a human 3D normal bronchial epithelial model (NHBE) under Air\u2013Liquid Interphase (ALI) and dendritic cells (DCs), tested in monoculture and in co-culture. Barrier function, measured by transepithelial electrical resistance (TEER), remained stable after 48\u00a0h of exposure to 400\u00a0nM siRNA RyR2, scramble control, or CaP NPs, indicating preserved epithelial performance. Confocal imaging showed efficient internalization of Cy5-labeled siRNA in both mono- and co-cultures. Cytokine profiling revealed IL-8 release across all conditions in NHBE and NHBE\u2009+\u2009DC models, with IL-6 and TNF-\u03b1 limited to immune-competent co-cultures; IFN-\u03b3 was below the limit of detection. No cytotoxicity was observed. Together, these data demonstrate that CaP NP\u2013mediated delivery achieves robust siRNA uptake without compromising airway barrier integrity, while eliciting only modest, context-dependent immune responses (primarily IL-8 and TNF-\u03b1 in the presence of DCs). This work supports the respiratory safety and translational potential of inhaled RyR2-targeting siRNA for CPVT.<\/jats:p>","DOI":"10.1007\/s00204-026-04370-7","type":"journal-article","created":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T09:51:28Z","timestamp":1776246688000},"page":"3053-3061","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Imaging and safety profiling of inhaled siRNA RyR2 in human respiratory models"],"prefix":"10.1007","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-5511-9520","authenticated-orcid":false,"given":"Valeria","family":"Bettinsoli","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdalena","family":"Erlacher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valentina","family":"Galbiati","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Marinovich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emanuela","family":"Corsini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5888-5118","authenticated-orcid":false,"given":"Doris","family":"Wilflingseder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,4,15]]},"reference":[{"key":"4370_CR1","doi-asserted-by":"publisher","first-page":"174178","DOI":"10.1016\/j.ejphar.2021.174178","volume":"905","author":"W Alshaer","year":"2021","unstructured":"Alshaer W, Zureigat H, Al Karaki A, Al-Kadash A, Gharaibeh L, Hatmal MM, Aljabali AAA, Awidi A (2021) siRNA: mechanism of action, challenges, and therapeutic approaches. Eur J Pharmacol 905:174178. https:\/\/doi.org\/10.1016\/j.ejphar.2021.174178","journal-title":"Eur J Pharmacol"},{"issue":"17","key":"4370_CR2","doi-asserted-by":"publisher","first-page":"5884","DOI":"10.1093\/nar\/gkq347","volume":"38","author":"BR Anderson","year":"2010","unstructured":"Anderson BR, Muramatsu H, Nallagatla SR, Bevilacqua PC, Sansing LH, Weissman D, Karik\u00f3 K (2010) Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res 38(17):5884\u20135892. https:\/\/doi.org\/10.1093\/nar\/gkq347","journal-title":"Nucleic Acids Res"},{"key":"4370_CR3","doi-asserted-by":"publisher","first-page":"422","DOI":"10.1007\/978-1-0716-2010-6","volume-title":"Antisense RNA design, delivery, and analysis","author":"V Arechavala-Gomeza","year":"2022","unstructured":"Arechavala-Gomeza V, Garanto A (2022) Antisense RNA design, delivery, and analysis. Springer Nature, p 422"},{"issue":"1","key":"4370_CR4","doi-asserted-by":"publisher","first-page":"L280","DOI":"10.1152\/ajplung.00122.2021","volume":"321","author":"CM Bartman","year":"2021","unstructured":"Bartman CM, Stelzig KE, Linden DR, Prakash YS, Chiarella SE (2021) Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures. Am J Physiol Lung Cell Mol Physiol 321(1):L280\u2013L286. https:\/\/doi.org\/10.1152\/ajplung.00122.2021","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"issue":"2","key":"4370_CR5","doi-asserted-by":"publisher","first-page":"180","DOI":"10.1007\/s10637-016-0407-y","volume":"35","author":"MS Beg","year":"2017","unstructured":"Beg MS, Brenner AJ, Sachdev J, Borad M, Kang YK, Stoudemire J, Smith S, Bader AG, Kim S, Hong DS (2017) Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients with advanced solid tumors. Invest New Drugs 35(2):180\u2013188. https:\/\/doi.org\/10.1007\/s10637-016-0407-y","journal-title":"Invest New Drugs"},{"issue":"6868","key":"4370_CR6","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1038\/415198a","volume":"415","author":"DM Bers","year":"2002","unstructured":"Bers DM (2002) Cardiac excitation-contraction coupling. Nature 415(6868):198\u2013205. https:\/\/doi.org\/10.1038\/415198a","journal-title":"Nature"},{"key":"4370_CR7","doi-asserted-by":"publisher","DOI":"10.3390\/life15010095","author":"V Bettinsoli","year":"2025","unstructured":"Bettinsoli V, Melzi G, Crea A, Degli Esposti L, Iafisco M, Catalucci D, Ciana P, Corsini E (2025) A novel approach for in vitro testing and hazard evaluation of nanoformulated RyR2-targeting siRNA drugs using human PBMCs. Life. https:\/\/doi.org\/10.3390\/life15010095","journal-title":"Life"},{"issue":"4","key":"4370_CR8","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1038\/s41573-023-00859-3","volume":"23","author":"D Bitounis","year":"2024","unstructured":"Bitounis D, Jacquinet E, Rogers MA, Amiji MM (2024) Strategies to reduce the risks of mRNA drug and vaccine toxicity. Nat Rev Drug Discov 23(4):281\u2013300. https:\/\/doi.org\/10.1038\/s41573-023-00859-3","journal-title":"Nat Rev Drug Discov"},{"issue":"5","key":"4370_CR9","doi-asserted-by":"publisher","first-page":"525","DOI":"10.1161\/CIRCRESAHA.117.310882","volume":"121","author":"R Bongianino","year":"2017","unstructured":"Bongianino R, Denegri M, Mazzanti A, Lodola F, Vollero A, Boncompagni S, Fasciano S, Rizzo G, Mangione D, Barbaro S, Di Fonso A, Napolitano C, Auricchio A, Protasi F, Priori SG (2017) Allele-specific silencing of mutant MRNA rescues ultrastructural and arrhythmic phenotype in mice carriers of the R4496C mutation in the Ryanodine Receptor Gene (RYR2). Circ Res 121(5):525\u2013536. https:\/\/doi.org\/10.1161\/CIRCRESAHA.117.310882","journal-title":"Circ Res"},{"issue":"8","key":"4370_CR10","doi-asserted-by":"publisher","first-page":"1223","DOI":"10.1080\/03639045.2018.1451879","volume":"44","author":"L Degli Esposti","year":"2018","unstructured":"Degli Esposti L, Carella F, Adamiano A, Tampieri A, Iafisco M (2018) Calcium Phosphate-based nanosystems for advanced targeted nanomedicine. Drug Dev Ind Pharm 44(8):1223\u20131238. https:\/\/doi.org\/10.1080\/03639045.2018.1451879","journal-title":"Drug Dev Ind Pharm"},{"issue":"8","key":"4370_CR11","doi-asserted-by":"publisher","first-page":"891","DOI":"10.2217\/nnm.16.26","volume":"11","author":"V Di Mauro","year":"2016","unstructured":"Di Mauro V, Iafisco M, Salvarani N, Vacchiano M, Carullo P, Ram\u00edrez-Rodr\u00edguez GB, Patr\u00edcio T, Tampieri A, Miragoli M, Catalucci D (2016) Bioinspired negatively charged calcium phosphate nanocarriers for cardiac delivery of microRNAs. Nanomedicine 11(8):891\u2013906. https:\/\/doi.org\/10.2217\/nnm.16.26","journal-title":"Nanomedicine"},{"key":"4370_CR12","doi-asserted-by":"publisher","unstructured":"Greeley MA (2017) Immunopathology of the respiratory system. Immunopathol Toxicol Drug Dev: Vol 2, Organ Syst 419\u2013453. https:\/\/doi.org\/10.1007\/978-3-319-47385-7_8","DOI":"10.1007\/978-3-319-47385-7_8"},{"issue":"1","key":"4370_CR13","doi-asserted-by":"publisher","first-page":"723","DOI":"10.1038\/s41467-018-02989-4","volume":"9","author":"MM Janas","year":"2018","unstructured":"Janas MM, Schlegel MK, Harbison CE, Yilmaz VO, Jiang Y, Parmar R, Zlatev I, Castoreno A, Xu H, Shulga-Morskaya S, Rajeev KG, Manoharan M, Keirstead ND, Maier MA, Jadhav V (2018) Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun 9(1):723. https:\/\/doi.org\/10.1038\/s41467-018-02989-4","journal-title":"Nat Commun"},{"issue":"11","key":"4370_CR14","doi-asserted-by":"publisher","first-page":"2704","DOI":"10.1007\/s13346-023-01361-4","volume":"13","author":"A Kaushalb","year":"2023","unstructured":"Kaushalb A (2023) Innate immune regulations and various SiRNA modalities. Drug Deliv Transl Res 13(11):2704\u20132718. https:\/\/doi.org\/10.1007\/s13346-023-01361-4","journal-title":"Drug Deliv Transl Res"},{"issue":"6","key":"4370_CR15","doi-asserted-by":"publisher","first-page":"L1068","DOI":"10.1152\/ajplung.00025.2008","volume":"294","author":"JL Koff","year":"2008","unstructured":"Koff JL, Shao MX, Ueki IF, Nadel JA (2008) Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium. Am J Physiol Lung Cell Mol Physiol 294(6):L1068\u2013L1075. https:\/\/doi.org\/10.1152\/ajplung.00025.2008","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"issue":"10","key":"4370_CR16","doi-asserted-by":"publisher","first-page":"2085","DOI":"10.1038\/s12276-023-01086-x","volume":"55","author":"Y Lee","year":"2023","unstructured":"Lee Y, Jeong M, Park J, Jung H, Lee H (2023) Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics. Exp Mol Med 55(10):2085\u20132096. https:\/\/doi.org\/10.1038\/s12276-023-01086-x","journal-title":"Exp Mol Med"},{"key":"4370_CR17","doi-asserted-by":"publisher","first-page":"331","DOI":"10.3389\/fimmu.2017.00331","volume":"8","author":"Z Meng","year":"2017","unstructured":"Meng Z, Lu M (2017) RNA interference-induced innate immunity, off-target effect, or immune adjuvant? Front Immunol 8:331. https:\/\/doi.org\/10.3389\/fimmu.2017.00331","journal-title":"Front Immunol"},{"issue":"1","key":"4370_CR18","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1038\/ncpcardio1399","volume":"6","author":"NL Mills","year":"2009","unstructured":"Mills NL, Donaldson K, Hadoke PW, Boon NA, MacNee W, Cassee FR, Sandstr\u00f6m T, Blomberg A, Newby DE (2009) Adverse cardiovascular effects of air pollution. Nat Clin Pract Cardiovasc Med 6(1):36\u201344. https:\/\/doi.org\/10.1038\/ncpcardio1399","journal-title":"Nat Clin Pract Cardiovasc Med"},{"issue":"424","key":"4370_CR19","doi-asserted-by":"publisher","first-page":"eaan6205","DOI":"10.1126\/scitranslmed.aan6205","volume":"10","author":"M Miragoli","year":"2018","unstructured":"Miragoli M, Ceriotti P, Iafisco M, Vacchiano M, Salvarani N, Alogna A, Carullo P, Ramirez-Rodr\u00edguez GB, Patr\u00edcio T, Esposti LD, Rossi F, Ravanetti F, Pinelli S, Alinovi R, Erreni M, Rossi S, Condorelli G, Post H, Tampieri A, Catalucci D (2018) Inhalation of peptide-loaded nanoparticles improves heart failure. Sci Transl Med 10(424):eaan6205. https:\/\/doi.org\/10.1126\/scitranslmed.aan6205","journal-title":"Sci Transl Med"},{"issue":"1","key":"4370_CR20","doi-asserted-by":"publisher","first-page":"385","DOI":"10.3390\/ijms22010385","volume":"22","author":"W Najahi-Missaoui","year":"2020","unstructured":"Najahi-Missaoui W, Arnold RD, Cummings BS (2020) Safe nanoparticles: Are we there yet? Int J Mol Sci 22(1):385. https:\/\/doi.org\/10.3390\/ijms22010385","journal-title":"Int J Mol Sci"},{"issue":"2","key":"4370_CR21","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1161\/01.cir.103.2.196","volume":"103","author":"SG Priori","year":"2001","unstructured":"Priori SG, Napolitano C, Tiso N, Memmi M, Vignati G, Bloise R, Sorrentino V, Danieli GA (2001) Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation 103(2):196\u2013200. https:\/\/doi.org\/10.1161\/01.cir.103.2.196","journal-title":"Circulation"},{"key":"4370_CR22","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1186\/s12989-014-0063-3","volume":"11","author":"M Savi","year":"2014","unstructured":"Savi M, Rossi S, Bocchi L, Gennaccaro L, Cacciani F, Perotti A, Amidani D, Alinovi R, Goldoni M, Aliatis I, Lottici P, Bersani D, Campanini M, Pinelli S, Petyx M, Frati C, Gervasi A, Urbanek K, Quaini F, Buschini A, Zaniboni M (2014) Titanium dioxide nanoparticles promote arrhythmias via a direct interaction with rat cardiac tissue. Part Fibre Toxicol 11:63. https:\/\/doi.org\/10.1186\/s12989-014-0063-3","journal-title":"Part Fibre Toxicol"},{"key":"4370_CR23","doi-asserted-by":"publisher","first-page":"788373","DOI":"10.3389\/fmats.2021.788373","volume":"8","author":"Z Sun","year":"2021","unstructured":"Sun Z, Li W, Lenzo JC, Holden JA, McCullough MJ, O\u2019Connor AJ, O\u2019Brien-Simpson NM (2021) The potential of Calcium Phosphate nanoparticles as adjuvants and vaccine delivery vehicles. Front Mater 8:788373. https:\/\/doi.org\/10.3389\/fmats.2021.788373","journal-title":"Front Mater"},{"issue":"3","key":"4370_CR24","doi-asserted-by":"publisher","first-page":"187","DOI":"10.1093\/rb\/rbw010","volume":"3","author":"X Xu","year":"2016","unstructured":"Xu X, Li Z, Zhao X, Keen L, Kong X (2016) Calcium phosphate nanoparticles-based systems for siRNA delivery. Regen Biomater 3(3):187\u2013195. https:\/\/doi.org\/10.1093\/rb\/rbw010","journal-title":"Regen Biomater"},{"issue":"4","key":"4370_CR25","doi-asserted-by":"publisher","first-page":"862","DOI":"10.1124\/pr.120.019554","volume":"72","author":"AM Yu","year":"2020","unstructured":"Yu AM, Choi YH, Tu MJ (2020) RNA drugs and RNA targets for small molecules: principles, progress, and challenges. Pharmacol Rev 72(4):862\u2013898. https:\/\/doi.org\/10.1124\/pr.120.019554","journal-title":"Pharmacol Rev"},{"issue":"7","key":"4370_CR26","doi-asserted-by":"publisher","first-page":"644","DOI":"10.1038\/s41419-022-05075-2","volume":"13","author":"Y Zhu","year":"2022","unstructured":"Zhu Y, Zhu L, Wang X, Jin H (2022) RNA-based therapeutics: an overview and prospectus. Cell Death Dis 13(7):644. https:\/\/doi.org\/10.1038\/s41419-022-05075-2","journal-title":"Cell Death Dis"}],"container-title":["Archives of Toxicology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00204-026-04370-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s00204-026-04370-7","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00204-026-04370-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T22:19:52Z","timestamp":1782512392000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s00204-026-04370-7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,15]]},"references-count":26,"journal-issue":{"issue":"7","published-print":{"date-parts":[[2026,7]]}},"alternative-id":["4370"],"URL":"https:\/\/doi.org\/10.1007\/s00204-026-04370-7","relation":{},"ISSN":["0340-5761","1432-0738"],"issn-type":[{"value":"0340-5761","type":"print"},{"value":"1432-0738","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,15]]},"assertion":[{"value":"28 January 2026","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"16 March 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"15 April 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}]}}