Points quantiques : caractérisation et applications en sciences pharmaceutiques

Medical imaging based on fluorescence has suffered from the poor photostability and mediocre performance of organic fluorophores. The discovery and subsequent improvements in nanocrystal synthesis and functionalization has greatly benefited the applications in medical imaging and the development of nanocrystal-based sensors for diagnostics. QDs are semi-conductor nanocrystals which have similar sizes as proteins (2-10 nm). They are highly luminescent, and can be made to emit at any desired wavelength by varying their size and composition. The surface of QDs can be easily functionalized with biomolecules. Hence, it is interesting to study how QDs interact in the biological world. Highly luminescent core-shell QDs emitting at different wavelengths were prepared according to our needs. In a first study, the surface of the QDs was modified with various small bi-functional thiolated ligands (carboxylated, aminated and zwitterionic). The modified-QDs of nearly identical sizes were administered in vitro to study the impact of surface charge and cell type on the mode and extent of cell uptake and elimination. Using specific inhibitors of cell uptake we determined which modes contributed to the internalization of the QDs. Endocytosis mediated by lipid rafts represented the predominant pathway for the internalization of QDs. However, other modes contributed to a lesser degree, depending on the surface ligand. We then analyzed the effect of QD agglomeration in cell culture media on its cellular uptake by microglia. Thorough characterization of QD agglomerate size distribution was conducted by asymmetrical flow field-flow fractionation (AF4) with a dynamic light scattering detector. Depending on the type of surface ligand and if serum proteins were present, the agglomeration pattern of the QDs was significantly different. With inhibitors of specific modes of cell uptake, we showed that the size distribution data, obtained by AF4, correlated with the modes of cell uptake. Microglia cells are immune cells of the central nervous system (CNS). They respond to injury or the presence of inflammagens by producing pro-inflammatory cytokine. Inflammation in the CNS may lead to loss of neurons, and can found in many chronic diseases. We were interested in building nanosensors to measure the onset of inflammation. Current methods to study inflammation consist in measuring levels of certain proteins or chemicals released by stressed cell (e.g. Western blot or ELISA assay for IL-1β). Although precise, these methods measure indirectly the activity of the enzyme responsible for releasing IL-1β, i.e. caspase-1. Moreover, these methods cannot be applied to live cells. We designed a sensor based on FRET between a QD and a dye linked by a peptide specifically cleaved by the caspase-1. To induce inflammation, we applied lipopolysaccharides (LPS), which are endotoxins present in Gram negative bacteria responsible for sceptic shock. The LPS form nanoparticles due to their amphiphilicity. The interior hydrophobic regions were used to load hydrophobic QDs, making the LPS luminescent. The microglia internalized LPS-QD predominantly through TLR-4 membrane receptors. We describe how the LPS induce inflammation and demonstrated the functionality of the QD-based sensor. Eventually, the sensor could be used to monitor in real time the action of therapeutics against inflammation.

Keyphrases: phagocytose, quantum dot, QD cell internalization, sub-cellular localization, asymmetrical flow field-flow fractionation, size distribution, cell culture media, serum, microglia, phagocytosis, nanosensor