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We previously demonstrated that impairment of the AIF\/CHCHD4-dependent protein import pathway disrupts mitochondrial bioenergetics and cell survival, highlighting this protein complex as a promising candidate for therapeutic intervention. Building on this concept, we identified a 27-residue peptide (N27) capable of perturbing the AIF-CHCHD4 interaction. However, its poor cellular uptake and inefficient mitochondrial localization have restricted its applicability. Here, we report on the development of a fluorescent nanodiamond (FND)-based nanosystem functionalized with a mitochondria-targeting peptide to transport and localize N27 at mitochondria. For this purpose, FNDs, as small as 40 nm, were doubly functionalized with polyethylene glycol (PEG) chains of two distinct lengths, providing covalent bonding for the mitochondria-targeting moiety and electrostatic interactions with the bioactive N27 peptide. FNDs were characterized before and after the addition of the peptides, with systematic assessment of the fraction of the bioactive peptide complexed as well as nanocarrier stability. Optimal conditions for copper-free click chemistry have also been defined in order to graft the mitochondria-targeting peptide without compromising the N27 loading or the stability of the nanosystem. Total internal reflection fluorescence and confocal microscopy demonstrated an increased cellular uptake of targeted FNDs. The FNDs colocalized with mitochondria, as well as the therapeutic peptide. This study provides the first description of dual-functionalized FNDs for mitochondria-targeted peptide delivery, establishing their potential as biocompatible and traceable nanoplatforms for subcellular delivery of bioactive peptides.<\/jats:p>","DOI":"10.1021\/acsabm.5c02455","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T04:23:15Z","timestamp":1788236595000},"page":"7916-7929","source":"Crossref","is-referenced-by-count":0,"title":["Fluorescent Nanodiamond-Based Delivery Systems for Mitochondrial Targeting of Bioactive Peptides"],"prefix":"10.1021","volume":"9","author":[{"given":"Dorna","family":"Akbarzadeh","sequence":"first","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, CNRS, Inserm, UTCBS , ,","place":["Paris, France, 75006"]},{"name":"Universit\u00e9 Paris-Saclay, CNRS, Gustave Roussy, METSY , ,","place":["Villejuif, France, 94805"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan Carlos","family":"Calder\u00f3n de la Rosa","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris-Saclay, CNRS, Gustave Roussy, METSY , ,","place":["Villejuif, France, 94805"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charles","family":"Fletcher","sequence":"additional","affiliation":[{"name":"Columbus NanoWorks, Inc. , , ,","place":["Columbus, Ohio, United States, 43212"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arfaan A.","family":"Rampersaud","sequence":"additional","affiliation":[{"name":"Columbus NanoWorks, Inc. , , ,","place":["Columbus, Ohio, United States, 43212"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marie-Odile","family":"David","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Evry Paris-Saclay, Inserm U1204, Laboratoire Structure et Activit\u00e9 des Biomol\u00e9cules Normales et Pathologiques , ,","place":["Evry, France, 91025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1755-0688","authenticated-orcid":true,"given":"Fran\u00e7ois","family":"Treussart","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris-Saclay, ENS Paris-Saclay, CNRS, CentraleSup\u00e9lec, LuMIn , ,","place":["Gif-sur-Yvette, France, 91190"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nazanine","family":"Modjtahedi","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris-Saclay, CNRS, Gustave Roussy, METSY , ,","place":["Villejuif, France, 94805"]},{"name":"Universit\u00e9 Claude Bernard Lyon1, UMR CNRS 5261, Inserm U1315, Institut NeuroMyoG\u00e8ne, Unit\u00e9 Physiopathologie et G\u00e9n\u00e9tique du Neurone et du Muscle , ,","place":["Lyon, France, 69008"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8390-4213","authenticated-orcid":true,"given":"Giorgia","family":"Urbinati","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris Cit\u00e9, CNRS, Inserm, UTCBS , ,","place":["Paris, France, 75006"]},{"name":"Universit\u00e9 Paris-Saclay, CNRS, Gustave Roussy, METSY , ,","place":["Villejuif, France, 94805"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,25]]},"reference":[{"issue":"6","key":"2026092215065927100_cit1","doi-asserted-by":"publisher","first-page":"1100","DOI":"10.1038\/s41591-022-01826-y","article-title":"Why drug delivery is the key to new medicines","volume":"28","author":"May","year":"2022","journal-title":"Nat. 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