{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T05:30:44Z","timestamp":1773207044252,"version":"3.50.1"},"reference-count":34,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2020,12,18]],"date-time":"2020-12-18T00:00:00Z","timestamp":1608249600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","award":["NMP-06-2015-683356 FOLSMART"],"award-info":[{"award-number":["NMP-06-2015-683356 FOLSMART"]}],"id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/04469\/2020"],"award-info":[{"award-number":["UIDB\/04469\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["SFRH\/BD\/140321\/2018"],"award-info":[{"award-number":["SFRH\/BD\/140321\/2018"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["SFRH\/BD\/121673\/2016"],"award-info":[{"award-number":["SFRH\/BD\/121673\/2016"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"name":"BioTecNorte","award":["NORTE-01-0145-FEDER-000004"],"award-info":[{"award-number":["NORTE-01-0145-FEDER-000004"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomedicines"],"abstract":"<jats:p>Methotrexate (MTX) is a common drug used to treat rheumatoid arthritis. Due to the excessive side effects, encapsulation of MTX in liposomes is considered an effective delivery system, reducing drug toxicity, while maintaining its efficacy. The ethanol injection method is an interesting technique for liposome production, due to its simplicity, fast implementation, and reproducibility. However, this method occasionally requires the extrusion process, to obtain suitable size distribution, and achieve a low level of MTX encapsulation. Here, we develop a novel pre-concentration method, based on the principles of the ethanol injection, using an initial aqueous volume of 20% and 1:1 ratio of organic:aqueous phase (v\/v). The liposomes obtained present small values of size and polydispersity index, without the extrusion process, and a higher MTX encapsulation (efficiency higher than 30%), suitable characteristics for in vivo application. The great potential of MTX to interact at the surface of the lipid bilayer was shown by nuclear magnetic resonance (NMR) studies, revealing mutual interactions between the drug and the main phospholipid via hydrogen bonding. In vivo experiments reveal that liposomes encapsulating MTX significantly increase the biological benefit in arthritic mice. This approach shows a significant advance in MTX therapeutic applications.<\/jats:p>","DOI":"10.3390\/biomedicines8120630","type":"journal-article","created":{"date-parts":[[2020,12,18]],"date-time":"2020-12-18T11:42:09Z","timestamp":1608291729000},"page":"630","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":48,"title":["Increased Encapsulation Efficiency of Methotrexate in Liposomes for Rheumatoid Arthritis Therapy"],"prefix":"10.3390","volume":"8","author":[{"given":"Diana","family":"Guimar\u00e3es","sequence":"first","affiliation":[{"name":"Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal"}]},{"given":"Jennifer","family":"Noro","sequence":"additional","affiliation":[{"name":"Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal"}]},{"given":"Ana","family":"Loureiro","sequence":"additional","affiliation":[{"name":"Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal"}]},{"given":"Franck","family":"Lager","sequence":"additional","affiliation":[{"name":"INSERM\u2014Institut National de la Sant\u00e9 et de la Recherche M\u00e9dicale, U1016, Institut Cochin, 75014 Paris, France"}]},{"given":"Gilles","family":"Renault","sequence":"additional","affiliation":[{"name":"INSERM\u2014Institut National de la Sant\u00e9 et de la Recherche M\u00e9dicale, U1016, Institut Cochin, 75014 Paris, France"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7204-2064","authenticated-orcid":false,"given":"Artur","family":"Cavaco-Paulo","sequence":"additional","affiliation":[{"name":"Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal"}]},{"given":"Eug\u00e9nia","family":"Nogueira","sequence":"additional","affiliation":[{"name":"Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal"},{"name":"Solfarcos\u2014Pharmaceutical and Cosmetic Solutions, 4710-053 Braga, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2020,12,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2757","DOI":"10.1002\/art.22056","article-title":"Tumor necrosis factor \u03b1 antagonist use and cancer in patients with rheumatoid arthritis","volume":"54","author":"Setoguchi","year":"2006","journal-title":"Arthritis Rheum."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1097\/BOR.0b013e328329c79d","article-title":"An update on methotrexate","volume":"21","author":"Braun","year":"2009","journal-title":"Curr. 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