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Here, we show that peptide architecture can influence intracellular trafficking and reduce accumulation within early endosomal compartments in lipid\u2013protein nanocarriers. Specifically, we fuse R6W3 (RRWWRRWRR), an amphipathic cell penetrating peptide, to the N- or C-terminus of the nanodisc scaffold proteins and systematically evaluate its impact on membrane interactions and cellular behavior. Structural and biophysical characterization confirms that R6W3 incorporation preserves nanodisc assembly and protein\u2013lipid interactions, enabling direct attribution of functional differences to peptide-driven interfacial effects. R6W3-functionalized nanodiscs exhibit enhanced membrane binding and cellular uptake, with N-terminal fusion producing the strongest interfacial interactions. In live cells, R6W3 functionalization increases endocytic activity, evidenced by increased formation of clathrin-coated pits and intracellular colocalization with clathrin-coated vesicles. Notably, R6W3-functionalized nanodiscs display reduced accumulation in early endosomes relative to unmodified nanodiscs, consistent with altered intracellular trafficking following endocytic uptake. Functionally, doxorubicin-loaded, R6W3-functionalized nanodiscs produced a greater reduction in cell viability than unmodified controls at equivalent concentrations. Together, these results demonstrate that R6W3 functionalization is an important design consideration for modulating nanodisc-cell interactions, from membrane association and cellular uptake to intracellular trafficking. These findings provide a framework for engineering nanodisc-based delivery systems with improved intracellular access.<\/jats:p>","DOI":"10.1021\/acsabm.6c01154","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T12:02:36Z","timestamp":1790337756000},"page":"9656-9669","source":"Crossref","is-referenced-by-count":0,"title":["Amphiphilic Peptide Fusion Promotes Endocytic Uptake of Nanodiscs"],"prefix":"10.1021","volume":"9","author":[{"given":"Brandon S.","family":"Pizarro Carbajal","sequence":"first","affiliation":[{"name":"University of Southern California , , , ,","place":["Los Angeles, California, United States, 90089"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trevin G.","family":"Reinhardt","sequence":"additional","affiliation":[{"name":"University of Southern California , , , ,","place":["Los Angeles, California, United States, 90089"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason A.","family":"Semenske","sequence":"additional","affiliation":[{"name":"R\u00edo Hondo College , , , ,","place":["Whittier, California, United States, 90601"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zekai","family":"Ji","sequence":"additional","affiliation":[{"name":"University of Southern California , , , ,","place":["Los Angeles, California, United States, 90089"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ciara O.","family":"Jacobs","sequence":"additional","affiliation":[{"name":"University of Southern California , , , ,","place":["Los Angeles, California, United States, 90089"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2341-8923","authenticated-orcid":true,"given":"Wade","family":"Zeno","sequence":"additional","affiliation":[{"name":"University of Southern California , , , ,","place":["Los Angeles, California, United States, 90089"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,25]]},"reference":[{"issue":"7","key":"2026101904074148100_cit1","doi-asserted-by":"publisher","DOI":"10.1002\/jex2.49","article-title":"Extracellular vesicles and lipoproteins - Smart messengers of blood cells in the circulation","volume":"1","author":"Nguyen","year":"2022","journal-title":"J. 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