Peptide-nanoparticle ligation mediated by cutinase fusion for the development of cancer cell-targeted nanoconjugates.
The relationship between the positioning of ligands on the surface of nanoparticles and the structural features of nanoconjugates has been underestimated for a long time, albeit of primary importance to promote specific biological recognition at the nanoscale. In particular, it has been formerly observed that a proper molecular orientation can play a crucial role, first optimizing ligand immobilization onto the nanoparticles and, second, improving the targeting efficiency of the nanoconjugates. In this work, we present a novel strategy to afford peptide-oriented ligation using genetically modified cutinase fusion proteins, which combines the presence of a site-directed "capture" module based on an enzymatic unit and a "targeting" moiety consisting of the ligand terminal end of a genetically encoded polypeptide chain. As an example, the oriented presentation of U11 peptide, a sequence specific for the recognition of urokinase plasminogen activator receptor (uPAR), was achieved by enzyme-mediated conjugation with an irreversible inhibitor of cutinase, an alkylphosphonate p-nitrophenol ester linker, covalently bound to the surface of iron oxide nanoparticles. The targeting efficiency of the resulting protein-nanoparticle conjugates was assessed using uPAR-positive breast cancer cells exploiting confocal laser scanning microscopy and quantitative fluorescence analysis of confocal images. Ultrastructural analysis of transmission electron micrographs provided evidence of a receptor-mediated pathway of endocytosis. Our results showed that, despite the small average number of targeting peptides presented on the nanoparticles, our ligand-oriented nanoconjugates proved to be very effective in selectively binding to uPAR and in promoting the uptake in uPAR-positive cancer cells.
†Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Elisabetta
E
Galbiati
†Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Marco
M
Cassani
‡Nerviano Medical Sciences s.r.l., viale Pasteur 10, 20014 Nerviano (MI), Italy.
Paolo
P
Verderio
†Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Enzo
E
Martegani
†Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Miriam
M
Colombo
†Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Paolo
P
Tortora
§Dipartimento di Scienze Biomediche e Cliniche "Luigi Sacco", Università di Milano, Ospedale L. Sacco, via G.B. Grassi 74, 20157 Milano, Italy.
Serena
S
Mazzucchelli
†Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Davide
D
Prosperi
D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis; D005290:Ferric Compounds / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D058726:Nanoconjugates / Q000737:chemistry* / Q000648:ultrastructure; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D009596:Nitrophenols / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D055293:Receptors, Urokinase Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship
D000970:Antineoplastic Agents; D005290:Ferric Compounds; D058726:Nanoconjugates; D009596:Nitrophenols; D010455:Peptides; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; C000499:ferric oxide; D002265:Carboxylic Ester Hydrolases; C019952:cutinase