{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T04:55:27Z","timestamp":1787460927568,"version":"build-2736575974"},"reference-count":46,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2020,3,12]],"date-time":"2020-03-12T00:00:00Z","timestamp":1583971200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005010","name":"Associazione Italiana per la Ricerca sul Cancro","doi-asserted-by":"publisher","award":["IG15216"],"award-info":[{"award-number":["IG15216"]}],"id":[{"id":"10.13039\/501100005010","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005010","name":"Associazione Italiana per la Ricerca sul Cancro","doi-asserted-by":"publisher","award":["IG17009"],"award-info":[{"award-number":["IG17009"]}],"id":[{"id":"10.13039\/501100005010","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100009880","name":"Regione Lazio","doi-asserted-by":"publisher","award":["85-\u00ad\u2010\u20112017-\u00ad\u2010\u201113750"],"award-info":[{"award-number":["85-\u00ad\u2010\u20112017-\u00ad\u2010\u201113750"]}],"id":[{"id":"10.13039\/501100009880","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Uncontrolled MAPK signaling is the main oncogenic driver in metastatic melanomas bearing mutations in BRAF kinase. These tumors are currently treated with the combination of BRAF\/MEK inhibitors (MAPKi), but this therapy is plagued by drug resistance. In this context we recently discovered that several microRNAs are involved in the development of drug resistance. In particular miR-204-5p and miR-199b-5p were found to function as antagonists of resistance because their enforced overexpression is able to inhibit melanoma cell growth in vitro either alone or in combination with MAPKi. However, the use of miRNAs in therapy is hampered by their rapid degradation in serum and biological fluids, as well as by the poor intracellular uptake. Here, we developed lipid nanoparticles (LNPs) encapsulating miR-204-5p, miR-199b-5p individually or in combination. We obtained LNPs with mean diameters &lt; 200 nm and high miRNA encapsulation efficiency. These formulations were tested in vitro on several melanoma cell lines sensitive to MAPKi or rendered drug resistant. Our results show that LNPs encapsulating combinations of the two oncosuppressor miRNAs are highly efficient in impairing melanoma cell proliferation and viability, affect key signaling pathways involved in melanoma cell survival, and potentiate the efficacy of drugs inhibiting BRAF and MEK. These results warrant further assessment of the anti-tumor efficacy of oncosuppressor miRNAs encapsulating LNPs in in vivo tumor models.<\/jats:p>","DOI":"10.3390\/ijms21061930","type":"journal-article","created":{"date-parts":[[2020,3,12]],"date-time":"2020-03-12T12:22:51Z","timestamp":1584015771000},"page":"1930","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["In Vitro Biophysical and Biological Characterization of Lipid Nanoparticles Co-Encapsulating Oncosuppressors miR-199b-5p and miR-204-5p as Potentiators of Target Therapy in Metastatic Melanoma"],"prefix":"10.3390","volume":"21","author":[{"given":"Luigi","family":"Fattore","sequence":"first","affiliation":[{"name":"Istituto Nazionale Tumori IRCCS, \"Fondazione G. Pascale\", 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Virginia","family":"Campani","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ciro Francesco","family":"Ruggiero","sequence":"additional","affiliation":[{"name":"IRCCS, Istituto Nazionale Tumori \u201cRegina Elena\u201d, Via Elio Chianesi 53, 00144 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valentina","family":"Salvati","sequence":"additional","affiliation":[{"name":"IRCCS, Istituto Nazionale Tumori \u201cRegina Elena\u201d, Via Elio Chianesi 53, 00144 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9218-6134","authenticated-orcid":false,"given":"Domenico","family":"Liguoro","sequence":"additional","affiliation":[{"name":"Department of Molecular and Clinical Medicine, University of Roma \u201cSapienza\u201d, 00185 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorena","family":"Scotti","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gerardo","family":"Botti","sequence":"additional","affiliation":[{"name":"Istituto Nazionale Tumori IRCCS, \"Fondazione G. Pascale\", 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paolo Antonio","family":"Ascierto","sequence":"additional","affiliation":[{"name":"Istituto Nazionale Tumori IRCCS, \"Fondazione G. Pascale\", 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rita","family":"Mancini","sequence":"additional","affiliation":[{"name":"Department of Molecular and Clinical Medicine, University of Roma \u201cSapienza\u201d, 00185 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1210-1269","authenticated-orcid":false,"given":"Giuseppe","family":"De Rosa","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2851-8605","authenticated-orcid":false,"given":"Gennaro","family":"Ciliberto","sequence":"additional","affiliation":[{"name":"IRCCS, Istituto Nazionale Tumori \u201cRegina Elena\u201d, Via Elio Chianesi 53, 00144 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,3,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"35","DOI":"10.2147\/ITT.S134842","article-title":"Melanoma treatment in review","volume":"7","author":"Domingues","year":"2018","journal-title":"ImmunoTargets Ther."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1007\/s00262-019-02311-1","article-title":"First-Line therapy-Stratified survival in BRAF-Mutant melanoma: A retrospective multicenter analysis","volume":"68","author":"Schilling","year":"2019","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Pelster, M.S., and Amaria, R.N. (2019). Combined targeted therapy and immunotherapy in melanoma: A review of the impact on the tumor microenvironment and outcomes of early clinical trials. Ther. Adv. Med Oncol., 11.","DOI":"10.1177\/1758835919830826"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1186\/s12967-015-0401-8","article-title":"Activating PIK3CA mutations coexist with BRAF or NRAS mutations in a limited fraction of melanomas","volume":"13","author":"Manca","year":"2015","journal-title":"J. Transl. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/S1470-2045(18)30142-6","article-title":"Encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF-Mutant melanoma (COLUMBUS): A multicentre, open-Label, randomised phase 3 trial","volume":"19","author":"Dummer","year":"2018","journal-title":"Lancet Oncol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1056\/NEJMoa1412690","article-title":"Improved overall survival in melanoma with combined dabrafenib and trametinib","volume":"372","author":"Robert","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1248","DOI":"10.1158\/2159-8290.CD-17-0401","article-title":"Recurrent Tumor Cell-Intrinsic and -Extrinsic Alterations during MAPKi-Induced Melanoma Regression and Early Adaptation","volume":"7","author":"Song","year":"2017","journal-title":"Cancer Discov."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e1405206","DOI":"10.1080\/2162402X.2017.1405206","article-title":"PD-L1 expression with immune-infiltrate evaluation and outcome prediction in melanoma patients treated with ipilimumab","volume":"7","author":"Madonna","year":"2018","journal-title":"Oncoimmunology"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1007\/s00262-018-2258-1","article-title":"Soluble CTLA-4 as a favorable predictive biomarker in metastatic melanoma patients treated with ipilimumab: An Italian melanoma intergroup study","volume":"68","author":"Pistillo","year":"2019","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1480","DOI":"10.1016\/S1470-2045(18)30700-9","article-title":"Nivolumab plus ipilimumab or nivolumab alone versus ipilimumab alone in advanced melanoma (CheckMate 067): 4-Year outcomes of a multicentre, randomised, phase 3 trial","volume":"19","author":"Hodi","year":"2018","journal-title":"Lancet Oncol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1016\/j.cell.2017.01.017","article-title":"Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy","volume":"168","author":"Sharma","year":"2017","journal-title":"Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1080\/14728222.2019.1554057","article-title":"The potential of BRAF-Associated non-Coding RNA as a therapeutic target in melanoma","volume":"23","author":"Fattore","year":"2019","journal-title":"Expert Opin. Ther. Targets"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/j.ccell.2014.11.018","article-title":"Tunable-Combinatorial mechanisms of acquired resistance limit the efficacy of BRAF\/MEK cotargeting but result in melanoma drug addiction","volume":"27","author":"Moriceau","year":"2015","journal-title":"Cancer Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1271","DOI":"10.1016\/j.cell.2015.07.061","article-title":"Non-Genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance","volume":"162","author":"Hugo","year":"2015","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.cytogfr.2017.05.003","article-title":"MicroRNA-Driven deregulation of cytokine expression helps development of drug resistance in metastatic melanoma","volume":"36","author":"Fattore","year":"2017","journal-title":"Cytokine Growth Factor Rev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1186\/s13046-018-0989-7","article-title":"Inhibition of Stearoyl-CoA desaturase 1 reverts BRAF and MEK inhibition-Induced selection of cancer stem cells in BRAF-Mutated melanoma","volume":"37","author":"Pisanu","year":"2018","journal-title":"J. Exp. Clin. Cancer Res. CR"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1186\/1479-5876-11-180","article-title":"Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF\/MEK inhibition and is abrogated by anti-ErbB3 antibodies","volume":"11","author":"Fattore","year":"2013","journal-title":"J. Transl. Med."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Ruggiero, C.F., Malpicci, D., Fattore, L., Madonna, G., Vanella, V., Mallardo, D., Liguoro, D., Salvati, V., Capone, M., and Bedogni, B. (2019). ErbB3 Phosphorylation as Central Event in Adaptive Resistance to Targeted Therapy in Metastatic Melanoma: Early Detection in CTCs during Therapy and Insights into Regulation by Autocrine Neuregulin. Cancers (Basel), 11.","DOI":"10.3390\/cancers11101425"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"24823","DOI":"10.18632\/oncotarget.4485","article-title":"Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF\/MEK inhibitors in melanoma","volume":"6","author":"Fattore","year":"2015","journal-title":"Oncotarget"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"22262","DOI":"10.18632\/oncotarget.14763","article-title":"MicroRNAs in melanoma development and resistance to target therapy","volume":"8","author":"Fattore","year":"2017","journal-title":"Oncotarget"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"E5005","DOI":"10.1073\/pnas.1607753113","article-title":"miR-579-3p controls melanoma progression and resistance to target therapy","volume":"113","author":"Fattore","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1267","DOI":"10.1038\/s41418-018-0205-5","article-title":"Reprogramming miRNAs global expression orchestrates development of drug resistance in BRAF-mutated melanoma","volume":"26","author":"Fattore","year":"2019","journal-title":"Cell Death Differ."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"478","DOI":"10.3389\/fgene.2019.00478","article-title":"The Potential for microRNA Therapeutics and Clinical Research","volume":"10","author":"Hanna","year":"2019","journal-title":"Front. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.addr.2014.05.009","article-title":"In vivo delivery of miRNAs for cancer therapy: Challenges and strategies","volume":"81","author":"Chen","year":"2015","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Di Martino, M.T., Campani, V., Misso, G., Gallo Cantafio, M.E., Gull\u00e0, A., Foresta, U., Guzzi, P.H., Castellano, M., Grimaldi, A., and Gigantino, V. (2014). In Vivo Activity of MiR-34a Mimics Delivered by Stable Nucleic Acid Lipid Particles (SNALPs) against Multiple Myeloma. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0090005"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1016\/S0005-2736(00)00343-6","article-title":"Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: Formation of novel small multilamellar vesicle structures","volume":"1510","author":"Semple","year":"2001","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1084","DOI":"10.1038\/s41565-019-0591-y","article-title":"The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-Based drugs","volume":"14","author":"Akinc","year":"2019","journal-title":"Nat. Nanotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Scognamiglio, I., Di Martino, M.T., Campani, V., Virgilio, A., Galeone, A., Gulla, A., Gallo Cantafio, M.E., Misso, G., Tagliaferri, P., and Tassone, P. (2014). Transferrin-Conjugated SNALPs encapsulating 2\u2019-O-Methylated miR-34a for the treatment of multiple myeloma. Biomed. Res. Int.","DOI":"10.1155\/2014\/217365"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Campani, V., Salzano, G., Lusa, S., and De Rosa, G. (2016). Lipid Nanovectors to Deliver RNA Oligonucleotides in Cancer. Nanomaterials (Basel Switz.), 6.","DOI":"10.3390\/nano6070131"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e423","DOI":"10.1038\/cddis.2012.160","article-title":"miR-204 targets Bcl-2 expression and enhances responsiveness of gastric cancer","volume":"3","author":"Sacconi","year":"2012","journal-title":"Cell Death Dis."},{"key":"ref_31","first-page":"4735","article-title":"MiR-199a inhibits the angiogenic potential of endometrial stromal cells under hypoxia by targeting HIF-1alpha\/VEGF pathway","volume":"8","author":"Dai","year":"2015","journal-title":"Int. J. Clin. Exp. Pathol."},{"key":"ref_32","first-page":"4179","article-title":"miR-204 enhances p27 mRNA stability by targeting Brd4 in head and neck squamous cell carcinoma","volume":"16","author":"Wang","year":"2018","journal-title":"Oncol. Lett."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.dnarep.2016.04.008","article-title":"Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage","volume":"42","author":"Karimian","year":"2016","journal-title":"DNA Repair"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1038\/s41419-019-2048-5","article-title":"Single cell analysis to dissect molecular heterogeneity and disease evolution in metastatic melanoma","volume":"10","author":"Fattore","year":"2019","journal-title":"Cell Death Dis."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/s13046-019-1490-7","article-title":"MiR-200c sensitizes Olaparib-Resistant ovarian cancer cells by targeting Neuropilin 1","volume":"39","author":"Vescarelli","year":"2020","journal-title":"J. Exp. Clin. Cancer Res. CR"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2980","DOI":"10.1016\/j.bbagen.2017.04.005","article-title":"Impact of BRAF kinase inhibitors on the miRNomes and transcriptomes of melanoma cells. Biochim","volume":"1861","author":"Kozar","year":"2017","journal-title":"Biophys. Acta. Gen. Subj."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1186\/s13046-019-1238-4","article-title":"miR-126-3p down-Regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A","volume":"38","author":"Caporali","year":"2019","journal-title":"J. Exp. Clin. Cancer Res. CR"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/0005-2736(94)90038-8","article-title":"Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-Containing liposomes","volume":"1190","author":"Litzinger","year":"1994","journal-title":"Biochim. Biophys. Acta (BBA)-Biomembr."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/0003-2697(80)90269-9","article-title":"Colorimetric determination of phospholipids with ammonium ferrothiocyanate","volume":"104","author":"Stewart","year":"1980","journal-title":"Anal. Biochem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"79342","DOI":"10.18632\/oncotarget.12688","article-title":"DNMT3B in vitro knocking-Down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation","volume":"7","author":"Megiorni","year":"2016","journal-title":"Oncotarget"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.fct.2014.11.025","article-title":"Toxicity of aflatoxin B1 towards the vitamin D receptor (VDR)","volume":"76","author":"Costanzo","year":"2015","journal-title":"Food Chem. Toxicol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"856","DOI":"10.3389\/fphar.2018.00856","article-title":"MMP-9 as a Candidate Marker of Response to BRAF Inhibitors in Melanoma Patients with BRAF(V600E) Mutation Detected in Circulating-Free DNA","volume":"9","author":"Salemi","year":"2018","journal-title":"Front. Pharmacol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"D\u2032Amici, S., Ceccarelli, S., Vescarelli, E., Romano, F., Frati, L., Marchese, C., and Angeloni, A. (2013). TNFa modulates Fibroblast Growth Factor Receptor 2 gene expression through the pRB\/E2F1 pathway: Identification of a non-Canonical E2F binding motif. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0061491"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1877","DOI":"10.1002\/jcp.29121","article-title":"CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions","volume":"235","author":"Bruschini","year":"2020","journal-title":"J. Cell. Physiol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"De Vitis, C., Corleone, G., Salvati, V., Ascenzi, F., Pallocca, M., De Nicola, F., Fanciulli, M., di Martino, S., Bruschini, S., and Napoli, C. (2019). B4GALT1 Is a New Candidate to Maintain the Stemness of Lung Cancer Stem Cells. J. Clin. Med., 8.","DOI":"10.3390\/jcm8111928"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1186\/s40425-019-0569-1","article-title":"Proteomic test for anti-PD-1 checkpoint blockade treatment of metastatic melanoma with and without BRAF mutations","volume":"7","author":"Ascierto","year":"2019","journal-title":"J. Immunother. Cancer"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/6\/1930\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:06:18Z","timestamp":1760173578000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/6\/1930"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,3,12]]},"references-count":46,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["ijms21061930"],"URL":"https:\/\/doi.org\/10.3390\/ijms21061930","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,3,12]]}}}