{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T07:39:23Z","timestamp":1792395563334,"version":"4.4.0"},"reference-count":49,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2026,10,18]],"date-time":"2026-10-18T00:00:00Z","timestamp":1792281600000},"content-version":"vor","delay-in-days":290,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T00:00:00Z","timestamp":1767225600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100008731","name":"Association Europ\u00e9enne contre les Leucodystrophies","doi-asserted-by":"publisher","award":["G.A. ELA n. 2019\u2013008I2AV1\u2013NanoERT"],"award-info":[{"award-number":["G.A. ELA n. 2019\u2013008I2AV1\u2013NanoERT"]}],"id":[{"id":"10.13039\/501100008731","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008731","name":"Association Europ\u00e9enne contre les Leucodystrophies","doi-asserted-by":"publisher","award":["ELA n. 2023-006C4"],"award-info":[{"award-number":["ELA n. 2023-006C4"]}],"id":[{"id":"10.13039\/501100008731","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004462","name":"Consiglio Nazionale delle Ricerche","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004462","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["International Journal of Biomaterials"],"published-print":{"date-parts":[[2026,1]]},"abstract":"<jats:p>Enzyme aggregation and polymer encapsulation are complementary strategies used to overcome the intrinsic instability and limited bioavailability of hydrophilic enzymes in medical uses. In this work, we report a systematic study of polymer\u2010encapsulated cross\u2010linked enzyme aggregates (pe\u2010CLEAs) as a hierarchical nanostructured platform for the stabilization and delivery of galactosylceramidase (GALC), a lysosomal enzyme used in the enzyme replacement therapy for Krabbe disease.<\/jats:p>\n                  <jats:p>\n                    GALC\u2010CLEAs were produced via cross\u2010linking and controlled precipitation, yielding stable aggregates with small hydrodynamic diameters (40\u201366\u2009nm) and narrow polydispersity index (PDI &lt;\u20090.1). Scaling up the synthesis by 50\u2010fold markedly enhanced total enzymatic output, reaching 1225.4\u2009\u00b1\u2009213.2\u2009U\u00b7mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    . GALC\u2010CLEAs were subsequently encapsulated within PLGA nanoparticles. Systematic screening of enzyme\u2010to\u2010polymer ratios revealed an optimal formulation at 75\u2009\u03bcg GALC per mg PLGA, displaying a maximum specific activity of 6.0\u2009\u00b1\u20091.3\u2009U\u00b7mg\n                    <jats:sup>\u22121<\/jats:sup>\n                    .\n                  <\/jats:p>\n                  <jats:p>\n                    Colloidal stability and retention assays showed that pe\u2010CLEAs preserved full catalytic activity and retained enzyme confinement over 30\u2009days at 4\u00b0C. At 37\u00b0C, pe\u2010CLEAs exhibited time\u2010dependent size dynamics while retaining substantial activity. In vitro evaluations in primary fibroblasts derived from Twitcher mice (a Krabbe disease model) demonstrated complete cytocompatibility up to 5.0\u2009mg\u00b7mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    and robust intracellular recovery of GALC activity across tested doses (0.5\u20135.0\u2009mg\u00b7mL\n                    <jats:sup>\u22121<\/jats:sup>\n                    ), outperforming equivalent doses of native enzyme.\n                  <\/jats:p>\n                  <jats:p>This work provides a comprehensive physicochemical and functional characterization of pe\u2010CLEAs, elucidating how hierarchical aggregation and polymer encapsulation can synergistically preserve stability and bioactivity. The design principles outlined here support the rational development of polymer\u2013enzyme nanocarriers for enzyme replacement therapy and related biomedical applications.<\/jats:p>","DOI":"10.1155\/ijbm\/8346433","type":"journal-article","created":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T06:41:13Z","timestamp":1792392073000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["PLGA\u2010Encapsulated Cross\u2010Linked GALC Aggregates as a Hierarchical Nanostructured Platform for Enzyme Stabilization and Delivery"],"prefix":"10.1155","volume":"2026","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4000-3973","authenticated-orcid":false,"given":"Laura","family":"Colagiorgio","sequence":"first","affiliation":[{"name":"Istituto Nanoscienze \u2013 CNR, NEST-SNS  Piazza San Silvestro 12 56126 Pisa Italy"},{"name":"Interdisciplinary Research Center \u201cHealth Science\u201d Scuola Superiore Sant\u2019Anna  Piazza Martiri Della Libert\u00e0, 33 56127 Pisa Italy  sssup.it"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5860-3141","authenticated-orcid":false,"given":"Mariacristina","family":"Gagliardi","sequence":"additional","affiliation":[{"name":"Istituto Nanoscienze \u2013 CNR, NEST-SNS  Piazza San Silvestro 12 56126 Pisa Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1838-9064","authenticated-orcid":false,"given":"Ambra Del","family":"Grosso","sequence":"additional","affiliation":[{"name":"Istituto Nanoscienze \u2013 CNR, NEST-SNS  Piazza San Silvestro 12 56126 Pisa Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3291-9009","authenticated-orcid":false,"given":"Sara","family":"Carpi","sequence":"additional","affiliation":[{"name":"Istituto Nanoscienze \u2013 CNR, NEST-SNS  Piazza San Silvestro 12 56126 Pisa Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miriam De","family":"Sarlo","sequence":"additional","affiliation":[{"name":"Istituto Nanoscienze \u2013 CNR, NEST-SNS  Piazza San Silvestro 12 56126 Pisa Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9688-2356","authenticated-orcid":false,"given":"Marco","family":"Cecchini","sequence":"additional","affiliation":[{"name":"Istituto Nanoscienze \u2013 CNR, NEST-SNS  Piazza San Silvestro 12 56126 Pisa Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,18]]},"reference":[{"key":"e_1_2_10_1_2","doi-asserted-by":"publisher","DOI":"10.3390\/catal15090891"},{"key":"e_1_2_10_2_2","doi-asserted-by":"publisher","DOI":"10.1002\/bab.2117"},{"key":"e_1_2_10_3_2","doi-asserted-by":"publisher","DOI":"10.1002\/fbe2.12105"},{"key":"e_1_2_10_4_2","doi-asserted-by":"publisher","DOI":"10.1007\/s11356-021-16764-4"},{"key":"e_1_2_10_5_2","doi-asserted-by":"publisher","DOI":"10.1007\/s12033-021-00396-7"},{"key":"e_1_2_10_6_2","doi-asserted-by":"publisher","DOI":"10.1111\/jocd.14673"},{"key":"e_1_2_10_7_2","doi-asserted-by":"publisher","DOI":"10.3390\/bios13040476"},{"key":"e_1_2_10_8_2","doi-asserted-by":"publisher","DOI":"10.4155\/bio-2023-0207"},{"key":"e_1_2_10_9_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms22179181"},{"key":"e_1_2_10_10_2","doi-asserted-by":"publisher","DOI":"10.2174\/0115734080273835231127045336"},{"key":"e_1_2_10_11_2","doi-asserted-by":"publisher","DOI":"10.20517\/rdodj.2022.09"},{"key":"e_1_2_10_12_2","doi-asserted-by":"publisher","DOI":"10.1186\/s11671-023-03823-7"},{"key":"e_1_2_10_13_2","doi-asserted-by":"publisher","DOI":"10.1007\/s11274-025-04568-4"},{"key":"e_1_2_10_14_2","doi-asserted-by":"publisher","DOI":"10.3390\/molecules27206861"},{"key":"e_1_2_10_15_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms24108593"},{"key":"e_1_2_10_16_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-023-00647-2"},{"key":"e_1_2_10_17_2","doi-asserted-by":"publisher","DOI":"10.1039\/d3cs01065a"},{"key":"e_1_2_10_18_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.3c00592"},{"key":"e_1_2_10_19_2","doi-asserted-by":"publisher","DOI":"10.1038\/s43246-025-00983-0"},{"key":"e_1_2_10_20_2","doi-asserted-by":"publisher","DOI":"10.1021\/acsomega.5c05589"},{"key":"e_1_2_10_21_2","doi-asserted-by":"publisher","DOI":"10.1002\/cite.202200167"},{"key":"e_1_2_10_22_2","doi-asserted-by":"publisher","DOI":"10.1111\/1750-3841.70606"},{"key":"e_1_2_10_23_2","article-title":"Enhancing Edible Bird Nest Cleaning Process by Immobilizing Cross\u2010Linked Enzyme Aggregates (Cleas) of Keratinase","author":"Ramli A. 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