{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T09:09:30Z","timestamp":1790240970702,"version":"4.1.0"},"reference-count":51,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T00:00:00Z","timestamp":1789862400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Methods Protoc.","MPs"],"abstract":"<jats:p>The 4T1 cell line is a widely used model for triple-negative breast cancer (TNBC) research due to its high metastatic potential and resemblance to clinical progression. However, its biological characteristics often limit the efficiency and reproducibility of conventional nucleic acid delivery protocols. Here, we present an optimized non-viral delivery framework for efficient cell-associated uptake in 4T1 cells. Five carrier systems\u2014Xfect RNA polymer (Xfect), polyethyleneimine (PEI), chitosan (CHI), and gold nanoparticle conjugates (AuPEI and AuCHI)\u2014were systematically evaluated using both standard (adherent) and reverse (suspension) delivery approaches. A fluorescently labeled oligonucleotide (5\u2032-TYE563) served as a reporter cargo at final concentrations of 5 nM and 10 nM, comparing complexation times of 15 s and 15 min. The proposed protocol introduces a rapid complexation strategy combined with reverse delivery, significantly reducing preparation time while enhancing cell-associated uptake. Under optimized conditions (10 nM, 15 s, reverse), Xfect achieved near-complete cellular uptake with minimal cytotoxicity. Crucially, the colocalization of dual-fluorescence signals enabled the simultaneous validation of metabolic viability and internalized cargo within a single field of view, bypassing the need for independent bulk cytotoxicity assays. This methodology provides a rapid, reproducible, and cost-effective framework to standardize early-stage delivery conditions for difficult-to-transfect cell lines such as 4T1.<\/jats:p>","DOI":"10.3390\/mps9050137","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T09:01:38Z","timestamp":1789981298000},"page":"137","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells"],"prefix":"10.3390","volume":"9","author":[{"given":"Andrea","family":"Gonz\u00e1lez-B\u00e1ez","sequence":"first","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Medicina Veterinaria y Zootecnia, San Nicol\u00e1s de los Garza 66054, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ra\u00fal","family":"Rangel-L\u00f3pez","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Ciencias Biol\u00f3gicas, San Nicol\u00e1s de los Garza 66455, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gustavo","family":"Hern\u00e1ndez-Vidal","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Medicina Veterinaria y Zootecnia, San Nicol\u00e1s de los Garza 66054, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Armando","family":"Trejo-Ch\u00e1vez","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Medicina Veterinaria y Zootecnia, San Nicol\u00e1s de los Garza 66054, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3096-8699","authenticated-orcid":false,"given":"Luis","family":"Rodr\u00edguez-Tovar","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Medicina Veterinaria y Zootecnia, San Nicol\u00e1s de los Garza 66054, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0852-8948","authenticated-orcid":false,"given":"Diana","family":"Z\u00e1rate-Trivi\u00f1o","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Ciencias Biol\u00f3gicas, San Nicol\u00e1s de los Garza 66455, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5446-1238","authenticated-orcid":false,"given":"Diana","family":"Caballero-Hernandez","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Ciencias Biol\u00f3gicas, San Nicol\u00e1s de los Garza 66455, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rodolfo Nino","family":"Fong","sequence":"additional","affiliation":[{"name":"Veterinary School, University of Long Island, New York, NY 11548, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1428-5217","authenticated-orcid":false,"given":"Gerardo","family":"M\u00e9ndez-Zamora","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Agronom\u00eda, San Nicol\u00e1s de los Garza 66054, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2225-0495","authenticated-orcid":false,"given":"Diana Elisa","family":"Zamora-Avila","sequence":"additional","affiliation":[{"name":"Universidad Aut\u00f3noma de Nuevo Le\u00f3n, Facultad de Medicina Veterinaria y Zootecnia, San Nicol\u00e1s de los Garza 66054, Nuevo L\u00e9on, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,20]]},"reference":[{"key":"ref_1","first-page":"229","article-title":"Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries","volume":"74","author":"Bray","year":"2024","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Zhu, L., Zhu, J., Wang, Q., Sun, X., Yuan, Y., Ding, S., Zhao, J., Liu, L., and Hou, S. (2025). Global, regional, and national burden of 34 cancer groups across 204 countries and territories, 1990\u20132021, and projections to 2050: A systematic analysis of the Global Burden of Disease Study 2021. Front. Oncol., 15.","DOI":"10.3389\/fonc.2025.1660125"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e70052","DOI":"10.1111\/1759-7714.70052","article-title":"Global, regional, and national burden of breast cancer, 1990\u20132021, and projections to 2050: A systematic analysis of the Global Burden of Disease Study 2021","volume":"16","author":"Deng","year":"2025","journal-title":"Thorac. Cancer"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e70044","DOI":"10.1002\/cbf.70044","article-title":"Application of gene editing in triple-negative breast cancer research","volume":"43","author":"Feng","year":"2025","journal-title":"Cell Biochem. Funct."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Rose, J.K. (2003). Optimization of transfection. Curr. Protoc. Cell Biol.","DOI":"10.1002\/0471143030.cb2007s19"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Khan, M. (2024). Polymers as efficient non-viral gene delivery vectors: The role of the chemical and physical architecture of mac-romolecules. Polymers, 16.","DOI":"10.3390\/polym16182629"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e00684","DOI":"10.1002\/asia.202500684","article-title":"Non-viral gene delivery systems: Current advances and therapeutic applications","volume":"20","author":"Wang","year":"2025","journal-title":"Chem. Asian J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3919","DOI":"10.1021\/jacs.4c12637","article-title":"Folding and functionalizing DNA origami: A versatile approach using a reactive polyamine","volume":"147","author":"Postigo","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"107332","DOI":"10.1016\/j.jddst.2025.107332","article-title":"Non-covalent modifications of DNA origami nanostructures to enhance drug delivery for breast cancer therapy","volume":"113","author":"Om","year":"2025","journal-title":"J. Drug Deliv. Sci. Technol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e11165","DOI":"10.7717\/peerj.11165","article-title":"Transfection types, methods and strategies: A technical review","volume":"9","author":"Chong","year":"2021","journal-title":"PeerJ"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Fus-Kujawa, A., Prus, P., Bajdak-Rusinek, K., Teper, P., Gawron, K., Kowalczuk, A., and Sieron, A.L. (2021). An overview of methods and tools for transfection of eukaryotic cells in vitro. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.701031"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"18102","DOI":"10.1039\/C5RA25333K","article-title":"Nanoparticles as transfection agents: A comprehensive study with ten different cell lines","volume":"6","author":"Neuhaus","year":"2016","journal-title":"RSC Adv."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Schr\u00f6rs, B., Boegel, S., Albrecht, C., Bukur, T., Bukur, V., Holtstr\u00e4ter, C., Ritzel, C., Manninen, K., Tadmor, A.D., and Vormehr, M. (2020). Multi-omics characterization of the 4T1 murine mammary gland tumor model. Front. Oncol., 10.","DOI":"10.3389\/fonc.2020.01195"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1016\/j.ejpb.2007.09.002","article-title":"Cell line-dependent internalization pathways and intracellular trafficking de-termine transfection efficiency of nanoparticle vectors","volume":"68","author":"Douglas","year":"2008","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Radzevi\u010di\u016bt\u0117, E., Maly\u0161ko-Pta\u0161insk\u0117, V., Novickij, J., Novickij, V., and Girkontait\u0117, I. (2022). Transfection by electroporation of cancer and primary cells using nanosecond and microsecond electric fields. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14061239"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3896","DOI":"10.3892\/ol.2016.5158","article-title":"RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo","volume":"12","author":"Liu","year":"2016","journal-title":"Oncol. Lett."},{"key":"ref_17","first-page":"799","article-title":"Optimization of transfection conditions for expression of green fluorescent protein in 4T1 mice mammary tumor cells","volume":"10","author":"Arora","year":"2021","journal-title":"Pharma Innov. J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1080\/10717544.2019.1709919","article-title":"Enhanced cytotoxicity of a redox-sensitive hyaluronic acid-based nano-medicine toward different oncocytes via various internalization mechanisms","volume":"27","author":"Du","year":"2020","journal-title":"Drug Deliv."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/j.neo.2015.08.005","article-title":"Metabolic plasticity of metastatic breast cancer cells: Adaptation to changes in the microenvironment","volume":"17","author":"Serganova","year":"2015","journal-title":"Neoplasia"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Teoh, S.T., Ogrodzinski, M.P., Ross, C., Hunter, K.W., and Lunt, S.Y. (2018). Sialic Acid Metabolism: A Key Player in Breast Cancer Me-tastasis Revealed by Metabolomics. Front. Oncol., 8.","DOI":"10.3389\/fonc.2018.00174"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e18208","DOI":"10.1002\/adma.202518208","article-title":"Stiffness-Gated Cytoplasmic mRNA Delivery Through Engineered Membrane Fusion for Breast Cancer Immunotherapy","volume":"38","author":"Chen","year":"2026","journal-title":"Adv. Mater."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e20240017","DOI":"10.1002\/smo.20240017","article-title":"Understanding of endo\/lysosomal escape of nanomaterials in biomedical application","volume":"3","author":"Wang","year":"2025","journal-title":"Smart Mol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1186\/s12967-025-07592-y","article-title":"Activation of the cGAS-STING pathway by clofoctol through mitochondrial damage in triple-negative breast cancer","volume":"24","author":"Wang","year":"2025","journal-title":"J. Transl. Med."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Reus, J.B., Trivino-Soto, G.S., Wu, L.I., Kokott, K., and Lim, E.S. (2020). SV40 Large T Antigen Is Not Responsible for the Loss of STING in 293T Cells but Can Inhibit cGAS-STING Interferon Induction. Viruses, 12.","DOI":"10.3390\/v12020137"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"6208","DOI":"10.1021\/jp0277107","article-title":"The Kinetics of DNA\u2212Cationic Vesicle Complex Formation","volume":"107","author":"Barreleiro","year":"2003","journal-title":"J. Phys. Chem. B"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Volovat, S.R., Augustin, I.G., Volovat, C., Vasilache, I., Ostafe, M., Panaite, D.I., Burlacu, A., and Volovat, C.C. (2026). Rewriting Tumor Entry Rules: Microfluidic Polyplexes and Tumor-Penetrating Strategies-A Literature Review. Pharmaceutics, 18.","DOI":"10.3390\/pharmaceutics18010084"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"239","DOI":"10.3892\/etm.2025.12989","article-title":"Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes","volume":"30","author":"Shimizu","year":"2025","journal-title":"Exp. Ther. Med."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Zheng, X., Li, R., Huang, H., Shen, J., Nie, W., Qiu, W., Chu, H., Qiao, Y., Li, Q., and Liu, H. (2022). Chitosan-Crosslinked Low Molecular Weight PEI-Conjugated Iron Oxide Nanoparticle for Safe and Effective DNA Delivery to Breast Cancer Cells. Nanomaterials, 12.","DOI":"10.3390\/nano12040584"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Karayianni, M., Sentoukas, T., Skandalis, A., Pippa, N., and Pispas, S. (2023). Chitosan-Based Nanoparticles for Nucleic Acid Delivery: Technological Aspects, Applications, and Future Perspectives. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15071849"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1038\/s41434-023-00386-1","article-title":"Reversible stabilization of DNA\/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity","volume":"30","author":"Maze","year":"2023","journal-title":"Gene Ther."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Migone, C., Fabiano, A., Zambito, Y., Piccarducci, R., Marchetti, L., Giacomelli, C., Martini, C., and Piras, A.M. (2025). Linear DNA\u2013Chitosan Nanoparticles: Formulation Challenges and Transfection Efficiency in Lung Cell Line. Appl. Biosci., 4.","DOI":"10.3390\/applbiosci4020029"},{"key":"ref_32","first-page":"393","article-title":"Discriminated properties of PEI functionalized gold nanoparticles (Au-PEIs) predetermined by synthetic routes of ligand exchange and reduction processes: Paths and Fates","volume":"1","author":"Cho","year":"2019","journal-title":"TechConnect Briefs"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1038\/s41538-019-0034-z","article-title":"Chitosan-capped gold nanoparticles for indicating temperature abuse in frozen stored products","volume":"3","author":"Mohan","year":"2019","journal-title":"npj Sci. Food"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"315602","DOI":"10.1088\/0957-4484\/20\/31\/315602","article-title":"The synthesis of biocompatible and SERS-active gold nanoparticles using chitosan","volume":"20","author":"Potara","year":"2009","journal-title":"Nanotechnology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e2964","DOI":"10.1002\/jgm.2964","article-title":"Polymer functionalized gold nanoparticles as nonviral gene delivery reagents","volume":"19","author":"Sebastian","year":"2017","journal-title":"J. Gene Med."},{"key":"ref_36","first-page":"e29","article-title":"Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions","volume":"2","author":"Miles","year":"2015","journal-title":"JBM"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"224001","DOI":"10.1088\/1361-6528\/aa6d15","article-title":"The impact of microfluidic mixing of triblock micelleplexes on in vitro\/in vivo gene silencing and intracellular trafficking","volume":"28","author":"Feldmann","year":"2017","journal-title":"Nanotechnology"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Shaabani, E., Sharifiaghdam, M., De Keersmaecker, H., De Rycke, R., De Smedt, S., Faridi-Majidi, R., Braeckmans, K., and Fraire, J.C. (2021). Layer by Layer Assembled Chitosan-Coated Gold Nanoparticles for Enhanced siRNA Delivery and Silencing. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22020831"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Riley, M.K., and Vermerris, W. (2017). Recent Advances in Nanomaterials for Gene Delivery-A Review. Nanomaterials, 7.","DOI":"10.3390\/nano7050094"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Antoniou, V., Mourelatou, E.A., Galatou, E., Avgoustakis, K., and Hatziantoniou, S. (2024). Gene Therapy with Chitosan Nanoparticles: Modern Formulation Strategies for Enhancing Cancer Cell Transfection. Pharmaceutics, 16.","DOI":"10.3390\/pharmaceutics16070868"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"105032","DOI":"10.1016\/j.chemphyslip.2020.105032","article-title":"Cationic lipids for gene delivery: Many players, one goal","volume":"235","author":"Ponti","year":"2021","journal-title":"Chem. Phys. Lipids"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Wang, T., Larcher, L.M., Ma, L., and Veedu, R.N. (2018). Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides. Molecules, 23.","DOI":"10.3390\/molecules23102564"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Ibrahim, B.O., Akere, T.H., Chakraborty, S., Valsami-Jones, E., and Ali-Boucetta, H. (2023). Gold nanoparticles induced size de-pendent cytotoxicity on human alveolar adenocarcinoma cells by inhibiting the ubiquitin proteasome system. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15020432"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"5641","DOI":"10.1021\/nn102228s","article-title":"Cellular response of polyvalent oligonucleotide-gold nanoparticle conjugates","volume":"4","author":"Massich","year":"2010","journal-title":"ACS Nano"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"4120","DOI":"10.1021\/acsanm.5c03906","article-title":"Deciphering gold nanoparticle-induced cy-totoxicity for safer nanomedicine applications: Exploring the impact of conjugation chemistry, functional groups, and metal core properties","volume":"8","author":"Ramakrishnan","year":"2025","journal-title":"ACS Appl. Nano Mater."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2.1.1","DOI":"10.1002\/0471142301.ns0201s50","article-title":"Fluorescence Microscopy: A Concise Guide to Current Imaging Methods","volume":"50","author":"Combs","year":"2010","journal-title":"Curr. Protoc. Neurosci."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1131","DOI":"10.1128\/CDLI.8.6.1131-1135.2001","article-title":"Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants","volume":"8","author":"Neri","year":"2001","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1002\/fft2.44","article-title":"Guidelines for cell viability assays","volume":"1","author":"Kamiloglu","year":"2020","journal-title":"Food Front."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1093\/jrr\/rru105","article-title":"Evaluation of chemical phototoxicity, focusing on phosphorylated histone H2AX","volume":"56","author":"Ibuki","year":"2015","journal-title":"J. Radiat. Res."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"294001","DOI":"10.1088\/1361-6463\/accc3d","article-title":"Phototoxicity and cell passage affect intra-cellular reactive oxygen species levels and sensitivity towards non-thermal plasma treatment in fluorescently-labeled cancer cells","volume":"56","author":"Verswyvel","year":"2023","journal-title":"J. Phys. D Appl. Phys."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Ghasemi, M., Turnbull, T., Sebastian, S., and Kempson, I. (2021). The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222312827"}],"container-title":["Methods and Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/5\/137\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T08:49:22Z","timestamp":1790239762000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-9279\/9\/5\/137"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,20]]},"references-count":51,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["mps9050137"],"URL":"https:\/\/doi.org\/10.3390\/mps9050137","relation":{},"ISSN":["2409-9279"],"issn-type":[{"value":"2409-9279","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,20]]}}}