NANOPARTICELLE SQUALENICHE AD ATTIVITà ANTIBIOTICA PER IL TRATTAMENTO DELLE INFEZIONI INTRACELLULARI

Several intracellular bacteria, such as Staphylococcus aureus, are able to survive and multiply within phagocytic cells, which become reservoirs of pathogenic bacteria giving rise to chronic infections. The treatment is difficult because most of available antibiotics, such as β-lactams, which are widely used for their low cost and side effects, have either poor intracellular diffusion and retention [1]. The aim of our project is to develop a colloidal system based on nanocarriers with high drug loading, to optimize the intracellular delivery of antibiotics. To this aim, we tried to use the squalenoylation platform [2] based on a biomimetic approach in which an endogenous steroid precursor, squalene (Sq), is able to adopt a compact conformation in aqueous medium. We have synthesized a new squalenic prodrug by esterification, penicillin G-squalene (SqPenG), which is able to form spontaneously in aqueous media stable nanoparticles (NPs) with a reduced average diameter and polydispersity. Remarkably, the NPs were shown to be stable in water during several weeks at different temperatures and concentrations, and also in biological media in the presence of serum. The morphology investigation carried out by CryoTEM and FFTEM shows a spherical shape with a full and amorphous supramolecular organization. The chemical stability and enzymatic degradation of SqPenG has been evaluated by 1H-NMR and reversed phase HPLC. Stealth® NPs of SqPenG to increase the blood half-life are obtained by co-nanoprecipitation or incorporation into preformed NPs of an amphiphilic PEG derivate of Sq (SqPEG). Fluorescent NPs have been obtained by co-nanoprecipitation of the hydrophobic green dye cholesteryl BODIPY® FL C12 in order to investigate and compare the intracellular delivery and trafficking of conventional and Stealth® NPs into macrophage cells by confocal microscopy. Indeed, the PEGylation decreases the rate of macrophages uptake but the NPs have the same internalization pathway and intracellular localization as compared to their non-PEGylated counterparts. The antibacterial activity of Sq-PenG NPs was then tested on Staphylococcus aureus-infected macrophages, using a Penicillin G-sensitive S. aureus strain. The squalenic prodrug showed a specific effect against intracellular bacteria and this activity is higher related to penicillin (‐50% of viable intracellular bacteria). Perspectives: 1) modification of the ester bond between squalene and penicillin G in order to increase the intracellular release of antibiotic; 2) the long life of Stealth® NPs will be evaluated in vivo by pharmacokinetic study and then PEG chains can be derivatized with ligands to give an active targeting to macrofages.