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Although the mechanisms underlying cell entry by similar peptides have been established to be temperature-dependent and cell-type specific and to involve receptor-mediated endocytosis, how these peptides cross the BBB remains unclear. Here, using an <jats:italic>in vitro<\/jats:italic> model, we studied the permeability of TAT, which was covalently bound to the fluorescent probe fluorescein isothiocyanate (FITC), and evaluated whether it crossed the \u201c<jats:italic>in vitro<\/jats:italic> BBB\u201d, a monolayer of brain endothelial cells, and whether the mechanisms were similar to those involved in TAT entry into cells. Our results show that although TAT crossed the monolayer of brain endothelial cells in a temperature-dependent manner, in contrast to the reported mechanism of cell entry, it did not require receptor-mediated endocytosis. Furthermore, we revisited the hypothesis that TAT facilitates brain delivery of covalently bound macromolecules by causing BBB disruption. Our results demonstrated that the dose of TAT commonly used in preclinical and clinical studies did not exert an effect on BBB permeability <jats:italic>in vitro<\/jats:italic> or <jats:italic>in vivo<\/jats:italic>; however, an extremely high TAT concentration caused BBB disruption <jats:italic>in vitro<\/jats:italic>. In conclusion, the BBB permeability to TAT is temperature-dependent, but at treatment-level concentrations, it does not involve receptor-mediated endocytosis or BBB disruption.<\/jats:p>","DOI":"10.1371\/journal.pone.0292681","type":"journal-article","created":{"date-parts":[[2023,10,11]],"date-time":"2023-10-11T17:31:25Z","timestamp":1697045485000},"page":"e0292681","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":12,"title":["TAT peptide at treatment-level concentrations crossed brain endothelial cell monolayer independent of receptor-mediated endocytosis or peptide-inflicted barrier disruption"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-1204-7887","authenticated-orcid":true,"given":"Meng-Chih","family":"Wu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric Yuhsiang","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6229-4134","authenticated-orcid":true,"given":"Ted Weita","family":"Lai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2023,10,11]]},"reference":[{"issue":"6","key":"pone.0292681.ref001","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1016\/0092-8674(88)90262-0","article-title":"Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein","volume":"55","author":"M Green","year":"1988","journal-title":"Cell"},{"issue":"6","key":"pone.0292681.ref002","doi-asserted-by":"crossref","first-page":"1189","DOI":"10.1016\/0092-8674(88)90263-2","article-title":"Cellular uptake of the tat protein from human immunodeficiency virus","volume":"55","author":"AD 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A reevaluation of the mechanism of cellular uptake","volume":"278","author":"JP Richard","year":"2003","journal-title":"The Journal of biological chemistry"},{"issue":"2","key":"pone.0292681.ref017","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1006\/bbrc.2002.6450","article-title":"Positively charged DNA-binding proteins cause apparent cell membrane translocation","volume":"291","author":"M Lundberg","year":"2002","journal-title":"Biochemical and biophysical research communications"},{"issue":"50","key":"pone.0292681.ref018","doi-asserted-by":"crossref","first-page":"50188","DOI":"10.1074\/jbc.M308719200","article-title":"Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells","volume":"278","author":"TB Potocky","year":"2003","journal-title":"The Journal of biological chemistry"},{"issue":"33","key":"pone.0292681.ref019","doi-asserted-by":"crossref","first-page":"31192","DOI":"10.1074\/jbc.M303938200","article-title":"Studies on the internalization mechanism of cationic cell-penetrating peptides","volume":"278","author":"G Drin","year":"2003","journal-title":"The Journal of biological chemistry"},{"issue":"2","key":"pone.0292681.ref020","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1016\/S1525-0016(03)00122-9","article-title":"Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time","volume":"8","author":"A Ferrari","year":"2003","journal-title":"Mol Ther"},{"issue":"36","key":"pone.0292681.ref021","doi-asserted-by":"crossref","first-page":"34141","DOI":"10.1074\/jbc.M303045200","article-title":"Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins","volume":"278","author":"A Fittipaldi","year":"2003","journal-title":"The Journal of biological chemistry"},{"issue":"42","key":"pone.0292681.ref022","doi-asserted-by":"crossref","first-page":"12652","DOI":"10.1021\/bi026097e","article-title":"Evidence for a plasma membrane-mediated permeability barrier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate","volume":"41","author":"S Violini","year":"2002","journal-title":"Biochemistry"},{"issue":"13","key":"pone.0292681.ref023","doi-asserted-by":"crossref","first-page":"12625","DOI":"10.1074\/jbc.M311461200","article-title":"A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides","volume":"279","author":"R Fischer","year":"2004","journal-title":"The Journal of biological chemistry"},{"issue":"1","key":"pone.0292681.ref024","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/S0006-291X(03)01135-5","article-title":"Uptake of analogs of penetratin, Tat(48\u201360) and oligoarginine in live cells","volume":"307","author":"PE Thoren","year":"2003","journal-title":"Biochemical and biophysical research communications"},{"issue":"3","key":"pone.0292681.ref025","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/nm996","article-title":"Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis","volume":"10","author":"JS Wadia","year":"2004","journal-title":"Nat Med"},{"issue":"6","key":"pone.0292681.ref026","doi-asserted-by":"crossref","first-page":"1011","DOI":"10.1016\/j.ymthe.2004.08.010","article-title":"Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement","volume":"10","author":"I Nakase","year":"2004","journal-title":"Mol Ther"},{"issue":"1","key":"pone.0292681.ref027","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/j.jconrel.2004.10.018","article-title":"Cationic TAT peptide transduction domain enters cells by macropinocytosis. 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Implications for their utilization as drug delivery vectors","volume":"17","author":"S Al-Taei","year":"2006","journal-title":"Bioconjug Chem"},{"issue":"2","key":"pone.0292681.ref035","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1042\/BJ20061808","article-title":"Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells","volume":"403","author":"MM Fretz","year":"2007","journal-title":"The Biochemical journal"},{"issue":"3","key":"pone.0292681.ref036","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1021\/bc700289w","article-title":"Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans","volume":"19","author":"M Kosuge","year":"2008","journal-title":"Bioconjug 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