2 research outputs found
Multilayered Magnetic Nanobeads for the Delivery of Peptides Molecules Triggered by Intracellular Proteases
In
this work, the versatility of layer-by-layer technology was combined
with the magnetic response of iron oxide nanobeads to prepare magnetic
mesostructures with a degradable multilayer shell into which a dye
quenched ovalbumin conjugate (DQ-OVA) was loaded. The system was specifically
designed to prove the protease sensitivity of the hybrid mesoscale
system and the easy detection of the ovalbumin released. The uptake
of the nanostructures in the breast cancer cells was followed by the
effective release of DQ-OVA upon activation via the intracellular
proteases degradation of the polymer shells. Monitoring the fluorescence
rising due to DQ-OVA digestion and the cellular dye distribution,
together with the electron microscopy studying, enabled us to track
the shell degradation and the endosomal uptake pathway that resulted
in the release of the digested fragments of DQ ovalbumin in the cytosol
Peptide-Nanoparticle Ligation Mediated by <i>Cutinase</i> 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 <i>cutinase</i> 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 <i>p</i>-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