Abstract
The fluorescent dye Cy3 and galactose derivatives are covalently assembled with different ratios on the surfaces of magnetic nanoparticles (MNPs) to produce multifunctional HepG2 cancer cell-targeting agents and the effect of ligand spatial orientation on the MNP surface is investigated on targeting specificity. By using a mixture of bis-N-hydroxysuccinimide ester (a bifunctional linker) and OSu-activated Cy3 (w/w = 30:1), stable and quantifiable fluorescent MNPs (Cy3@MNPs) are synthesized that could be subsequently loaded with galactosyl ligands. A mono-antennary and two different tri-antennary galactosyl ligands are individually immobilized on Cy3@MNPs, and the uptake efficiencies of the resulting galactosyl Cy3@MNPs by HepG2 and HeLa cells are investigated using confocal microscopy. The confocal images show that galactosyl Cy3@MNPs are sprayed over cytoplasm of the HepG2 cells, indicating that the MNP uptake occurs via receptor-mediated endocytosis that is followed by release from endosomes. The results also reveal that the ligand spatial orientation affects the efficiency of the receptor-mediated endocytosis and one of the tri-antennary galactosyl ligands shows the best uptake efficiency owing to the optimal spatial presentation of the galactosyl moieties. Overall, it is shown that the MNP is a good ligand carrier and that, when pre-assembled, the multivalent ligand structure enhances the interactions between the surface ligands of the MNPs and receptors of HepG2 cells. Additionally, the galactosyl Cy3@MNPs are not cytotoxic, indicating that they may potentially be used for in vivo applications. A multifunctional magnetic nanoparticle (MNP) with fluorescent, magnetic, and targeting properties is shown to be specifically ingested by HepG2 cells that abundantly express asialoglycoprotein receptors (ASGP-R) on their surfaces. The spatial orientation of the pre-assembled multivalent galactosyl ligand influences the MNP uptake by the HepG2 cell. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.