{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T04:19:52Z","timestamp":1771042792051,"version":"3.50.1"},"reference-count":53,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2021,3,4]],"date-time":"2021-03-04T00:00:00Z","timestamp":1614816000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/04138\/2020; UIDP\/04138\/2020; PTDC\/QUI\/64023\/2006; PTDC\/EBB-BIO\/101237\/2008; SFRH\/BSAB\/1210\/2011; SFRH\/BD\/47946\/2008"],"award-info":[{"award-number":["UIDB\/04138\/2020; UIDP\/04138\/2020; PTDC\/QUI\/64023\/2006; PTDC\/EBB-BIO\/101237\/2008; SFRH\/BSAB\/1210\/2011; SFRH\/BD\/47946\/2008"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009422","name":"National PKU Alliance","doi-asserted-by":"publisher","award":["Human phenylalanine hydroxylase and nanobiomaterials: a novel enzyme reposition therapy approach to PKU"],"award-info":[{"award-number":["Human phenylalanine hydroxylase and nanobiomaterials: a novel enzyme reposition therapy approach to PKU"]}],"id":[{"id":"10.13039\/100009422","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.<\/jats:p>","DOI":"10.3390\/pharmaceutics13030329","type":"journal-article","created":{"date-parts":[[2021,3,5]],"date-time":"2021-03-05T00:39:07Z","timestamp":1614904747000},"page":"329","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic Enzyme"],"prefix":"10.3390","volume":"13","author":[{"given":"Paulo R.","family":"Lino","sequence":"first","affiliation":[{"name":"Faculty of Pharmacy, Research Institute for Medicines, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]},{"given":"Jo\u00e3o","family":"Leandro","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Research Institute for Medicines, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]},{"given":"Mariana","family":"Amaro","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Research Institute for Medicines, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6799-2740","authenticated-orcid":false,"given":"L\u00eddia M. D.","family":"Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Research Institute for Medicines, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7807-4726","authenticated-orcid":false,"given":"Paula","family":"Leandro","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Research Institute for Medicines, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2946-9342","authenticated-orcid":false,"given":"Ant\u00f3nio J.","family":"Almeida","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Research Institute for Medicines, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Usmani, S.S., Bedi, G., Samuel, J.S., Singh, S., Kalra, S., Kumar, P., Ahuja, A.A., Sharma, M., Gautam, A., and Raghava, G.P.S. 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