{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:55:26Z","timestamp":1760144126853,"version":"build-2065373602"},"reference-count":49,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T00:00:00Z","timestamp":1711324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004837","name":"Ministerio de Ciencia e Innovaci\u00f3n","doi-asserted-by":"publisher","award":["PDI2021-122271OB-I00"],"award-info":[{"award-number":["PDI2021-122271OB-I00"]}],"id":[{"id":"10.13039\/501100004837","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Transfection agents play a crucial role in facilitating the uptake of nucleic acids into eukaryotic cells offering potential therapeutic solutions for genetic disorders. However, progress in this field needs the development of improved systems that guarantee efficient transfection. Here, we describe the synthesis of a set of chemical delivery agents (TRIFAPYs) containing alkyl chains of different lengths based on the 1,3,5-tris[(4-alkyloxy-1pyridinio)methyl]benzene tribromide structure. Their delivery properties for therapeutic oligonucleotides were evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs) as a silencing tool. The binding of liposomes to PPRHs was evaluated by retardation assays in agarose gels. The complexes had a size of 125 nm as determined by DLS, forming well-defined concentrical vesicles as visualized by Cryo-TEM. The prostate cancer cell line PC-3 was used to study the internalization of the nanoparticles by fluorescence microscopy and flow cytometry. The mechanism of entrance involved in the cellular uptake was mainly by clathrin-mediated endocytosis. Cytotoxicity analyses determined the intrinsic toxicity caused by each TRIFAPY and the effect on cell viability upon transfection of a specific PPRH (HpsPr-C) directed against the antiapoptotic target survivin. TRIFAPYs C12-C18 were selected to expand these studies in the breast cancer cell line SKBR-3 opening the usage of TRIFAPYs for both sexes and, in the hCMEC\/D3 cell line, as a model for the blood\u2013brain barrier. The mRNA levels of survivin decreased, while apoptosis levels increased upon the transfection of HpsPr-C with these TRIFAPYs in PC-3 cells. Therefore, TRIFAPYs can be considered novel lipid-based vehicles for the delivery of therapeutic oligonucleotides.<\/jats:p>","DOI":"10.3390\/biom14040390","type":"journal-article","created":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T12:47:11Z","timestamp":1711370831000},"page":"390","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Synthesis and Validation of TRIFAPYs as a Family of Transfection Agents for Therapeutic Oligonucleotides"],"prefix":"10.3390","volume":"14","author":[{"given":"Berta","family":"Isanta","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Toxicology and Therapeutic Chemistry, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5044-1762","authenticated-orcid":false,"given":"Ana","family":"Delgado","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7855-392X","authenticated-orcid":false,"given":"Carlos J.","family":"Ciudad","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"},{"name":"Nanoscience and Nanotechnology Institute, IN2UB, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6232-9886","authenticated-orcid":false,"given":"M\u00aa Ant\u00f2nia","family":"Busquets","sequence":"additional","affiliation":[{"name":"Nanoscience and Nanotechnology Institute, IN2UB, University of Barcelona, 08028 Barcelona, Spain"},{"name":"Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8747-1993","authenticated-orcid":false,"given":"Rosa","family":"Griera","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Toxicology and Therapeutic Chemistry, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"},{"name":"Nanoscience and Nanotechnology Institute, IN2UB, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6608-8872","authenticated-orcid":false,"given":"N\u00faria","family":"Llor","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Toxicology and Therapeutic Chemistry, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"},{"name":"Nanoscience and Nanotechnology Institute, IN2UB, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7937-8038","authenticated-orcid":false,"given":"V\u00e9ronique","family":"No\u00e9","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"},{"name":"Nanoscience and Nanotechnology Institute, IN2UB, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,3,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/2052-8426-2-27","article-title":"Gene Therapy for Cancer: Present Status and Future Perspective","volume":"2","author":"Amer","year":"2014","journal-title":"Mol. Cell. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1038\/nrd1775","article-title":"Design and Development of Polymers for Gene Delivery","volume":"4","author":"Pack","year":"2005","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1208\/ps040312","article-title":"Transfection Efficiency and Toxicity of Polyethylenimine in Differentiated Calu-3 and Nondifferentiated COS-1 Cell Cultures","volume":"4","author":"Florea","year":"2002","journal-title":"AAPS PharmSci"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Nayerossadat, N., Maedeh, T., and Ali, P. (2012). Viral and Nonviral Delivery Systems for Gene Delivery. Adv. Biomed. Res., 1.","DOI":"10.4103\/2277-9175.98152"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Lundstrom, K. (2018). Viral Vectors in Gene Therapy. Diseases, 6.","DOI":"10.3390\/diseases6020042"},{"key":"ref_6","unstructured":"Alsaggar, M., and Liu, D. (2015). Advances in Genetics, Elsevier."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"640","DOI":"10.1016\/j.copbio.2010.07.003","article-title":"Chemical Vectors for Gene Delivery: Uptake and Intracellular Trafficking","volume":"21","author":"Pichon","year":"2010","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1016\/j.actbio.2016.05.040","article-title":"Synthesis and Application of Poly(Ethylene Glycol)-Co-Poly(\u03b2-Amino Ester) Copolymers for Small Cell Lung Cancer Gene Therapy","volume":"41","author":"Kim","year":"2016","journal-title":"Acta Biomater."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1370","DOI":"10.1002\/biot.201400841","article-title":"Synthetic Vectors for Gene Delivery: An Overview of Their Evolution Depending on Routes of Administration","volume":"10","author":"Belmadi","year":"2015","journal-title":"Biotechnol. J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"7909","DOI":"10.1016\/j.biomaterials.2014.05.066","article-title":"Gene Transfection in Complex Media Using PCBMAEE-PCBMA Copolymer with Both Hydrolytic and Zwitterionic Blocks","volume":"35","author":"Zhang","year":"2014","journal-title":"Biomaterials"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Garc\u00eda-Garrido, E., Cordani, M., and Somoza, \u00c1. (2021). Modified Gold Nanoparticles to Overcome the Chemoresistance to Gemcitabine in Mutant P53 Cancer Cells. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13122067"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/j.ejpb.2021.05.016","article-title":"Synthesis and Validation of DOPY: A New Gemini Dioleylbispyridinium Based Amphiphile for Nucleic Acid Transfection","volume":"165","author":"Aubets","year":"2021","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Delgado, A., Griera, R., Llor, N., L\u00f3pez-Aguilar, E., Busquets, M.A., No\u00e9, V., and Ciudad, C.J. (2023). Trioleyl Pyridinium, a Cationic Transfection Agent for the Lipofection of Therapeutic Oligonucleotides into Mammalian Cells. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15020420"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"999","DOI":"10.1038\/sj.gt.3302024","article-title":"Gene Therapy Progress and Prospects: Gene Therapy for the Hemophilias","volume":"10","author":"Walsh","year":"2003","journal-title":"Gene Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1016\/j.gde.2020.05.022","article-title":"Nucleic Acid Therapeutics in Neurodevelopmental Disease","volume":"65","author":"Winkelsas","year":"2020","journal-title":"Curr. Opin. Genet. Dev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"457","DOI":"10.3390\/genes4030457","article-title":"Antisense Gene Silencing: Therapy for Neurodegenerative Disorders?","volume":"4","author":"Nielsen","year":"2013","journal-title":"Genes"},{"key":"ref_17","unstructured":"Highleyman, L. (1998). FDA Approves Fomivirsen, Famciclovir, and Thalidomide. Food and Drug Administration, BETA."},{"key":"ref_18","unstructured":"(2016). FDA Grants Accelerated Approval to First Drug for Duchenne Muscular Dystrophy. Case Med. Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1038\/s41573-020-0075-7","article-title":"Advances in Oligonucleotide Drug Delivery","volume":"19","author":"Roberts","year":"2020","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1421","DOI":"10.1517\/14656566.6.8.1421","article-title":"Pegaptanib: The First Antiangiogenic Agent Approved for Neovascular Macular Degeneration","volume":"6","author":"Doggrell","year":"2005","journal-title":"Expert. Opin. Pharmacother."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"No\u00e9, V., Aubets, E., F\u00e9lix, A.J., and Ciudad, C.J. (2021). Nucleic Acids Therapeutics Using PolyPurine Reverse Hoogsteen Hairpins. Biochem. Pharmacol., 189.","DOI":"10.1016\/j.bcp.2020.114371"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2809","DOI":"10.2174\/0929867324666170301114127","article-title":"Polypurine Reverse Hoogsteen Hairpins as a Gene Silencing Tool for Cancer","volume":"24","author":"Ciudad","year":"2017","journal-title":"Curr. Med. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1021\/mp5007008","article-title":"Improved Design of PPRHs for Gene Silencing","volume":"12","author":"Villalobos","year":"2015","journal-title":"Mol. Pharm."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"11579","DOI":"10.1074\/jbc.M900981200","article-title":"Polypurine Hairpins Directed against the Template Strand of DNA Knock Down the Expression of Mammalian Genes","volume":"284","author":"Coma","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1021\/mp400431f","article-title":"Stability and Immunogenicity Properties of the Gene-Silencing Polypurine Reverse Hoogsteen Hairpins","volume":"11","author":"Villalobos","year":"2014","journal-title":"Mol. Pharm."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.bcp.2018.06.020","article-title":"Functional Pharmacogenomics and Toxicity of PolyPurine Reverse Hoogsteen Hairpins Directed against Survivin in Human Cells","volume":"155","author":"Ciudad","year":"2018","journal-title":"Biochem. Pharmacol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Aubets, E., F\u00e9lix, A.J., Garav\u00eds, M., Reyes, L., Avi\u00f1\u00f3, A., Eritja, R., Ciudad, C.J., and No\u00e9, V. (2020). Detection of a G-Quadruplex as a Regulatory Element in Thymidylate Synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21145028"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1541","DOI":"10.1016\/j.bcp.2013.09.013","article-title":"Polypurine Reverse Hoogsteen Hairpins as a Gene Therapy Tool against Survivin in Human Prostate Cancer PC3 Cells In Vitro and In Vivo","volume":"86","author":"Villalobos","year":"2013","journal-title":"Biochem. Pharmacol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"9681","DOI":"10.1002\/chem.200901475","article-title":"Selective Formation of a Single Atropisomer of Meso\u2014Meso-Linked Zn II Diporphyrin through Supramolecular Self-Assembly","volume":"15","author":"Maeda","year":"2009","journal-title":"Chem.\u2014A Eur. J."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1038\/s41467-023-37113-8","article-title":"Poly(Heptazine Imide) Ligand Exchange Enables Remarkable Low Catalyst Loadings in Heterogeneous Metallaphotocatalysis","volume":"14","author":"Xing","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4202","DOI":"10.1021\/acs.iecr.2c00129","article-title":"Hydrolyzable Quaternary Pyridinium Surfactants: Antimicrobial Profragrances for Controllable Perfume Release","volume":"61","author":"Zhou","year":"2022","journal-title":"Ind. Eng. Chem. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1080\/026782998206434","article-title":"Preliminary Communication Properties of Nonlinear Supramolecular Liquid Crystals Containing Thiophenedicarboxylic Acids","volume":"25","author":"Lin","year":"1998","journal-title":"Liq. Cryst."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1089\/oli.2006.16.196","article-title":"A Web-Based Search Engine for Triplex-Forming Oligonucleotide Target Sequences","volume":"16","author":"Gaddis","year":"2006","journal-title":"Oligonucleotides"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1089\/1545457041526290","article-title":"Use of SiRNAs and Antisense Oligonucleotides Against Survivin RNA to Inhibit Steps Leading to Tumor Angiogenesis","volume":"14","author":"Coma","year":"2004","journal-title":"Oligonucleotides"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1016\/j.devcel.2006.04.002","article-title":"Dynasore, a Cell-Permeable Inhibitor of Dynamin","volume":"10","author":"Macia","year":"2006","journal-title":"Dev. Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1038\/mt.2009.281","article-title":"The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers: Optimization and Pitfalls","volume":"18","author":"Vercauteren","year":"2010","journal-title":"Mol. Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3640","DOI":"10.1111\/bph.14429","article-title":"Identification of Novel Macropinocytosis Inhibitors Using a Rational Screen of Food and Drug Administration-approved Drugs","volume":"175","author":"Lin","year":"2018","journal-title":"Br. J. Pharmacol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/S0928-0987(02)00280-4","article-title":"Physicochemical and Biological Evaluation of Cationic Polymethacrylates as Vectors for Gene Delivery","volume":"18","author":"Dubruel","year":"2003","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3062","DOI":"10.1039\/c3nj00272a","article-title":"Gene Delivery Agents Possessing Antiradical Activity: Self-Assembling Cationic Amphiphilic 1,4-Dihydropyridine Derivatives","volume":"37","author":"Pajuste","year":"2013","journal-title":"New J. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"104724","DOI":"10.1016\/j.jff.2021.104724","article-title":"Effect of Medium Chain Fatty Acid in Human Health and Disease","volume":"87","author":"Roopashree","year":"2021","journal-title":"J. Funct. Foods"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1186\/2045-8118-10-16","article-title":"The HCMEC\/D3 Cell Line as a Model of the Human Blood Brain Barrier","volume":"10","author":"Weksler","year":"2013","journal-title":"Fluids Barriers CNS"},{"key":"ref_42","unstructured":"Falsini, S., and Ristori, S. (2016). Non-Viral Gene Delivery Vectors, Humana Press."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1007\/s10120-003-0255-2","article-title":"Correlation between Survivin MRNA Expression and Lymph Node Metastasis in Gastric Cancer","volume":"6","author":"Miyachi","year":"2003","journal-title":"Gastric Cancer"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"957","DOI":"10.1097\/01.meg.0000230086.83792.56","article-title":"Expression of Survivin in Gastric Cancer and Its Relationship with Tumor Angiogenesis","volume":"18","author":"Lee","year":"2006","journal-title":"Eur. J. Gastroenterol. Hepatol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.1016\/j.ijrobp.2009.09.007","article-title":"Survivin Is a Potential Mediator of Prostate Cancer Metastasis","volume":"78","author":"Zhang","year":"2010","journal-title":"Int. J. Radiat. Oncol. Biol. Phys."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1186\/1746-1596-7-52","article-title":"Survivin Regulates the Expression of VEGF-C in Lymphatic Metastasis of Breast Cancer","volume":"7","author":"Cai","year":"2012","journal-title":"Diagn. Pathol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1248\/bpb.28.565","article-title":"Significance of Survivin MRNA Expression in Prognosis of Neuroblastoma","volume":"28","author":"Ito","year":"2005","journal-title":"Biol. Pharm. Bull."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1785","DOI":"10.1053\/jpsu.2001.28839","article-title":"The Inhibitor of Apoptosis Protein Survivin Is Associated with High-Risk Behavior of Neuroblastoma","volume":"36","author":"Azuhata","year":"2001","journal-title":"J. Pediatr. Surg."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2577","DOI":"10.3892\/ol.2014.2556","article-title":"Survivin and Vascular Endothelial Growth Factor Are Associated with Spontaneous Pulmonary Metastasis of Osteosarcoma: Development of an Orthotopic Mouse Model","volume":"8","author":"Zhang","year":"2014","journal-title":"Oncol. Lett."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/14\/4\/390\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:18:10Z","timestamp":1760105890000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/14\/4\/390"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,3,25]]},"references-count":49,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["biom14040390"],"URL":"https:\/\/doi.org\/10.3390\/biom14040390","relation":{},"ISSN":["2218-273X"],"issn-type":[{"type":"electronic","value":"2218-273X"}],"subject":[],"published":{"date-parts":[[2024,3,25]]}}}