{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T13:38:53Z","timestamp":1765373933163,"version":"3.46.0"},"reference-count":40,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T00:00:00Z","timestamp":1765238400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Vitarka Therapeutics Ltd."}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background: To address hepatotropic body distribution and toxicity, transfection systems based on protein architecture have been proposed. Attenuated anthrax toxin (aATx) has provided the backbone for a first in class transfection system that, in the wild, uses intraluminal vesicles (ILVs) as an intermediary compartment during the translocation of large molecules into the cytosol. Small interfering (si)RNA molecules non-covalently attached to a carrier (LFn-PKR) would not be predicted to be an aATx translocase (protective antigen (PA)) substrate. Previously, siRNA has been shown to be delivered to the cytosol using this system. Methods: Here, the localisation of 32P-labelled siRNA delivered using aATx was quantified directly and related to siRNA activity. In addition, inhibition of ILV formation by hypertonic sucrose or wheatgerm agglutinin (WGA) was shown to inhibit the aATx-mediated cytosolic translocation of siRNA. Results: MCF-7 cells were used to establish siRNA intracellular distribution in relation to pharmacological activity by targeting STAT3 gene expression. After Lipofectamine-mediated transfection using 100 nM 32P-labelled siRNA, 45 \u00b1 3.2% (\u00b1SD; n = 3) of the cell associated siRNA was found in the cytosol. After the transfection of 100 nM 32P-labelled siRNA using aATx, 77 \u00b1 2.5% (\u00b1SD; n = 3) of the cell associated siRNA was found in the cytosol and resulted in a reduction in STAT3 expression of 64.04 \u00b1 14.17% (\u00b1SD; n = 3) relative to an untreated control by Western analysis. Further, 25 \u03bcg\/mL of WGA inhibited 75.23 \u00b1 0.06% (\u00b1SD; n = 3) of the knockdown attributed to a non-WGA-treated control. Relative to the control, treatment with 200 mM sucrose resulted in a reduction of 74.58 \u00b1 7.76% (\u00b1SD; n = 3) of target gene knockdown. Conclusions: These data indicated that the insertion of the PA pore into endosomal membrane did not weaken the endosomal limiting membrane, leading to vesicular bursting during transfection and ILVs played critical role in translocase activity.<\/jats:p>","DOI":"10.3390\/pharmaceutics17121584","type":"journal-article","created":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T12:42:43Z","timestamp":1765284163000},"page":"1584","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Intraluminal Vesicles as Transfection Intermediaries"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6935-6222","authenticated-orcid":false,"given":"Nourhan A. M.","family":"Mahmoud","sequence":"first","affiliation":[{"name":"The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK"},{"name":"Department of Biochemistry, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Giza 12451, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hadeer K. S.","family":"Abdelrahman","sequence":"additional","affiliation":[{"name":"The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK"},{"name":"Department of Biochemistry, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Giza 12451, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benedita K. L.","family":"Feron","sequence":"additional","affiliation":[{"name":"The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK"},{"name":"Department of Chemistry, Ume\u00e5 University, 901 87 Ume\u00e5, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andra","family":"Pintilie","sequence":"additional","affiliation":[{"name":"The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marc","family":"Fivaz","sequence":"additional","affiliation":[{"name":"Remynd, Bio-Incubator, Gaston Geenslaan 1, 3001 Leuven, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joanna J.","family":"Miest-Bray","sequence":"additional","affiliation":[{"name":"The School of Sciences, Canterbury Christ Church University, Anselm Building, North Holmes Road, Canterbury, Kent CT1 1QU, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Timothy","family":"Gomez","sequence":"additional","affiliation":[{"name":"The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natalie","family":"Youens","sequence":"additional","affiliation":[{"name":"The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vineeta","family":"Tripathi","sequence":"additional","affiliation":[{"name":"Vitarka Therapeutics Ltd., Innovation House, Discovery Park Sandwich, Kent CT13 9FF, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7927-0649","authenticated-orcid":false,"given":"Simon C. W.","family":"Richardson","sequence":"additional","affiliation":[{"name":"The Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e048025","DOI":"10.1136\/bmjopen-2020-048025","article-title":"Correlates of intended COVID-19 vaccine acceptance across time and countries: Results from a series of cross-sectional surveys","volume":"11","author":"Kerr","year":"2021","journal-title":"BMJ Open"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Jo, S.J., Chae, S.U., Bin Lee, C., and Bae, S.K. (2023). Clinical Pharmacokinetics of Approved RNA Therapeutics. Int. J. Mol. 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