RESUMO
We report on a simple robust procedure for synthesis of generation-4 poly-(amidoamine) (PAMAM) dendrimers with a precisely core positioned single sulforhodamine B molecule. The labelled dendrimers exhibited high fluorescent quantum yields where the absorbance and fluorescence spectrum of the fluorophore was not affected by pH and temperature. Since the stoichiometry of the fluorophore to the dendrimer is 1:1, we were able to directly compare uptake kinetics, the mode of uptake, trafficking and safety of dendrimers of different end-terminal functionality (carboxylated vs. pyrrolidonated) by two phenotypically different human endothelial cell types (the human brain capillary endothelial cell line hCMEC/D3 and human umbilical vein endothelial cells), and without interference of the fluorophore in uptake processes. The results demonstrate comparable uptake kinetics and a predominantly clathrin-mediated endocytotic mechanism, irrespective of dendrimer end-terminal functionality, where the majority of dendrimers are directed to the endo-lysosomal compartments in both cell types. A minor fraction of dendrimers, however, localize to endoplasmic reticulum and the Golgi apparatus, presumably through the recycling endosomes. In contrast to amino-terminated PAMAM dendrimers, we confirm safety of carboxylic acid- and pyrrolidone-terminated PAMAM dendrimers through determination of cell membrane integrity and comprehensive respiratory profiling (measurements of mitochondrial oxidative phosphorylation and determination of its coupling efficiency). Our dendrimer core-labelling approach could provide a new conceptual basis for improved understanding of dendrimer performance within biological settings.
Assuntos
Dendrímeros/análise , Células Endoteliais/citologia , Corantes Fluorescentes/análise , Rodaminas/análise , Linhagem Celular , Dendrímeros/química , Dendrímeros/metabolismo , Células Endoteliais/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaboloma , Imagem Óptica , Rodaminas/química , Rodaminas/metabolismoRESUMO
A series of different generation PAMAM dendrimers with sulforhodamine B covalently attached to the dendrimer core was investigated regarding their optical properties. Steady-state and time-resolved spectroscopic techniques were used to determine the size influence of the dendrimers on the photophysical behavior of the luminescent core. New blue emissive species were formed as the generation increased from zero to four. The growth of the dendritic branches resulted in a rise of fluorescence quantum yield and fluorescence lifetime values. Rotational correlation times were used to determine the hydrodynamic diameters of the fluorescent-core dendrimers, and good accordance was found with the values previously reported for unlabeled PAMAM dendrimers, which makes them potentially suitable diagnostic tools for biomedical tracing.