{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,21]],"date-time":"2026-03-21T13:37:02Z","timestamp":1774100222592,"version":"3.50.1"},"reference-count":48,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T00:00:00Z","timestamp":1627257600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009422","name":"National PKU Alliance","doi-asserted-by":"publisher","award":["-"],"award-info":[{"award-number":["-"]}],"id":[{"id":"10.13039\/100009422","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\/04138\/2020, UIDP\/04138\/2020, PTDC\/QUI\/64023\/2006, PTDC\/EBB-BIO\/101237\/2008, SFRH\/BSAB\/1210\/2011 (to A.J.A.), SFRH\/BD\/47946\/2008 (to P.R.L.)"],"award-info":[{"award-number":["UIDB\/04138\/2020, UIDP\/04138\/2020, PTDC\/QUI\/64023\/2006, PTDC\/EBB-BIO\/101237\/2008, SFRH\/BSAB\/1210\/2011 (to A.J.A.), SFRH\/BD\/47946\/2008 (to P.R.L.)"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.<\/jats:p>","DOI":"10.3390\/ijms22157987","type":"journal-article","created":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T22:22:46Z","timestamp":1627338166000},"page":"7987","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles"],"prefix":"10.3390","volume":"22","author":[{"given":"Paulo R.","family":"Lino","sequence":"first","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal"}]},{"given":"Jo\u00e3o","family":"Leandro","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal"}]},{"given":"Lara","family":"Figueiredo","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal"}]},{"given":"Mariana P.","family":"Amaro","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 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":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2946-9342","authenticated-orcid":false,"given":"Paula","family":"Leandro","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7807-4726","authenticated-orcid":false,"given":"Ant\u00f3nio J.","family":"Almeida","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"118505","DOI":"10.1016\/j.ijpharm.2019.118505","article-title":"Science and art of protein formulation development","volume":"568","author":"Wang","year":"2019","journal-title":"Int. J. Pharm."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1637","DOI":"10.1016\/j.xphs.2018.12.014","article-title":"Immunogenicity of protein pharmaceuticals","volume":"108","author":"Dingman","year":"2019","journal-title":"J. Pharm. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1700513","DOI":"10.1002\/advs.201700513","article-title":"Polysaccharide-based controlled release systems for therapeutics delivery and tissue engineering: From bench to bedside","volume":"5","author":"Miao","year":"2018","journal-title":"Adv. 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