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Strategies involving the targeted delivery of antibiotics have been proposed to minimize the administered antibiotic doses. This study aims to develop the first double-modified nanovehicle capable of increasing bacterial membranes\u2019 permeability while specifically targeting Staphylococcus aureus, one of the foremost pathogens responsible for global mortality rates. Thus, polymeric NPs composed of poly(lactic-co-glycolic acid) (PLGA) were produced, and their surface was modified with TAT peptide to increase the membranes\u2019 permeability and folic acid (FA) to direct the NPs to S. aureus. The nanosystem showed spherical morphology with sizes of 174 \u00b1 4 nm, a monodisperse population (polydispersity index of 0.08 \u00b1 0.02), and a zeta potential of \u22122.5 \u00b1 0.1 mV. The NPs remained stable for up to four months during storage. Fluorescence-based flow cytometry analysis proved that the double modification of PLGA NPs increased the interaction of the NPs with S. aureus, with fluorescence increasing from 71 \u00b1 3% to 87 \u00b1 1%. The nanosystem slightly affected the growth curve of S. aureus by extending both the lag time (from 2.5 \u00b1 0.2 to 2.88 \u00b1 0.4 h) and the exponential phase, as evidenced by an increase in the half-maximum growth time (from 3.9 \u00b1 0.2 to 4.4 \u00b1 0.1 h). Furthermore, the nanocarrier showed no toxicity for human dermal fibroblast cells, maintaining a 100% cell viability at the highest concentration tested (100 \u00b5M). Therefore, the proposed FA\/TAT-functionalized nanocarrier presented promising features to be successfully used as a delivery vehicle of antimicrobials to fight S. aureus.<\/jats:p>","DOI":"10.3390\/ijms262110666","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:15:14Z","timestamp":1762175714000},"page":"10666","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["PLGA Nanoparticles Double-Decorated with a TAT Peptide and Folic Acid to Target Staphylococcus aureus"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6918-8775","authenticated-orcid":false,"given":"St\u00e9phanie","family":"Andrade","sequence":"first","affiliation":[{"name":"LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2428-7520","authenticated-orcid":false,"given":"Maria J.","family":"Ramalho","sequence":"additional","affiliation":[{"name":"LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"given":"Jo\u00e3o","family":"Santos","sequence":"additional","affiliation":[{"name":"LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"given":"S\u00edlvio","family":"Santos","sequence":"additional","affiliation":[{"name":"CEB\u2014Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal"},{"name":"LABBELS\u2014Associate Laboratory, University of Minho, 4710-057 Braga, Portugal"}]},{"given":"Lu\u00eds D. R.","family":"Melo","sequence":"additional","affiliation":[{"name":"CEB\u2014Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal"},{"name":"LABBELS\u2014Associate Laboratory, University of Minho, 4710-057 Braga, Portugal"},{"name":"Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7623-3877","authenticated-orcid":false,"given":"Nuno","family":"Guimar\u00e3es","sequence":"additional","affiliation":[{"name":"LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0274-106X","authenticated-orcid":false,"given":"Maria P.","family":"Ferraz","sequence":"additional","affiliation":[{"name":"Department of Mechanical Engineering, Faculty of Engineering, University of Porto, 4200-465 Porto, Portugal"},{"name":"i3S\u2014Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto, Portugal"},{"name":"INEB\u2014Institute of Biomedical Engineering, University of Porto, 4200-135 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5864-3250","authenticated-orcid":false,"given":"Nuno F.","family":"Azevedo","sequence":"additional","affiliation":[{"name":"LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8505-3432","authenticated-orcid":false,"given":"Maria C.","family":"Pereira","sequence":"additional","affiliation":[{"name":"LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9841-3967","authenticated-orcid":false,"given":"Joana A.","family":"Loureiro","sequence":"additional","affiliation":[{"name":"LEPABE, ALiCE, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"Department of Mechanical Engineering, Faculty of Engineering, University of Porto, 4200-465 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2221","DOI":"10.1016\/S0140-6736(22)02185-7","article-title":"Global mortality associated with 33 bacterial pathogens in 2019: A systematic analysis for the global burden of disease study 2019","volume":"400","author":"Ikuta","year":"2022","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Balloux, F., and van Dorp, L. 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