Transferrin receptor-1 iron-acquisition pathway - synthesis, kinetics, thermodynamics and rapid cellular internalization of a holotransferrin-maghemite nanoparticle construct.
2013
Abstract Background Targeting nanoobjects via the iron-acquisition pathway is always reported slower than the transferrin/receptor endocytosis. Is there a remedy? Methods Maghemite superparamagnetic and theragnostic nanoparticles (diameter 8.6 nm) were synthesized, coated with 3-aminopropyltriethoxysilane (NP) and coupled to four holotransferrin (TFe 2 ) by amide bonds (TFe 2 –NP). The constructs were characterized by X-ray diffraction, transmission electron microscopy, FTIR, X-ray Electron Spectroscopy, Inductively Coupled Plasma with Atomic Emission Spectrometry. The in-vitro protein/protein interaction of TFe 2 –NP with transferrin receptor-1 (R1) and endocytosis in HeLa cells were investigated spectrophotometrically, by fast T-jump kinetics and confocal microscopy. Results In-vitro, R1 interacts with TFe 2 –NP with an overall dissociation constant K D = 11 nM. This interaction occurs in two steps: in the first, the C-lobe of the TFe 2 –NP interacts with R1 in 50 μs: second-order rate constant, k 1 = 6 × 10 10 M − 1 s − 1 ; first-order rate constant, k − 1 = 9 × 10 4 s − 1 ; dissociation constant, K 1d = 1.5 μM. In the second step, the protein/protein adduct undergoes a slow (10,000 s) change in conformation to reach equilibrium. This mechanism is identical to that occurring with the free TFe 2 . In HeLa cells, TFe 2 –NP is internalized in the cytosol in less than 15 min. Conclusion This is the first time that a nanoparticle–transferrin construct is shown to interact with R1 and is internalized in time scales similar to those of the free holotransferrin. General significance TFe 2 –NP behaves as free TFe 2 and constitutes a model for rapidly targeting theragnostic devices via the main iron-acquisition pathway.
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