Fioretto, Laura (2020) Glycolipids with immunomodulant activity and their development as immunotherapy adjuvants and antitumor. [Tesi di dottorato]

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Abstract

Despite of the enormous efforts put in the research of new vaccines and vaccine adjuvants, approved formulations are far to be efficient against challenging diseases. In this frame, the development of new molecular vaccine adjuvants represents the most innovative technology in the identification of new vaccines. Inspired by a natural immunogenic α-sulfoquinovosyldiacylglycerols (α-SQDGs), this PhD project was aimed to the identification of glycolipids with immunomodulant activity and their development as vaccine adjuvants. The first synthetized sulfolipid was a β-SQDGs (1,2-O-distearoyl-3-O-β-D-sulfoquinovosylglycerol) named Sulfavant A. Sulfavant A presented the sulfonic group typical of natural α-SQDGs and the β anomeric configuration usual for LPS-based glycolipids. Sulfavant A activated dendritic cells (DC) with up-regulation of the CD83 as well as the gene expression of proinflammatory cytokines suggesting an interesting immunomodulant activity TLR-independent. Sulfavant A was also tested in in vivo: in a first experiment the antibodies response was evaluated, and the results were comparable to commercial adjuvants; in a second experiment the adjuvant properties of Sulfavant A were evaluated in a model of vaccination against melanoma in mice obtaining promising results. Starting from these results we explored the effect of structural changing on sulfolipids bioactivity introducing different acyl chains and modifying the stereochemistry at glyceridic carbon 2. In particular, two palmitoyl and two nonadecanoyl chains were introduced and for each molecule the epimeric mixture at glyceridic carbon 2 and the diasteropure analogues were synthetized. The biological assays suggested that even slight modifications in the hydrophobic part induced partial loss of activity, independently from the stereochemistry of the glyceridic carbon 2. The synthetic strategy was improved and employed for the preparation of the two diasteropure analogues of Sulfavant A, 1,2-O-distearoyl-3-O-β-D-sulfoquinovosyl-(R)-glycerol (named Sulfavant R) and 1,2-O-distearoyl-3-O-β-D-sulfoquinovosyl-(S)-glycerol (named Sulfavant S), with defined stereochemistry at carbon 2. The biological evaluation of Sulfavant R and Sulfavant S bioactivity led to different response correlated with stereochemical aspects; in fact, efficacy of single epimers (Sulfavant S and R) on DCs maturation was maximum at concentration 1000-fold lower than that occurred for the Sulfavant A while all the activity curves were featured by a peculiar bell-shaped dose response. A hypothesis of this different behaviour was that Sulfavants could self-assemble in aqueous environment influencing the bioavailability of the active molecule to the cellular target. The self-assembling properties of Sulfavants was investigated in this PhD thesis. In particular, Dynamic Light Scattering (DLS) gave the first evidence that Sulfavants aggregate in water forming supramolecular structure of different size. Despite some difficulties to analyse Sulfavants solutions at very low concentrations, the Critical Micellar Concentration (CMC) was evaluated by surface tension measurements and fluorescence spectroscopy. The assessment of the effect of a detergent on Sulfavant A showed that aggregation negatively affected the immunomodulant activity, since with the detergent the activation of DCs underwent shift at lower concentrations. Cryo-TEM images showed direct evidences of Sulfavant A and Sulfavant R aggregation at 10 nM and 10 µM concentrations, underlining the differences between the supramolecular structures at different concentrations at which they were able to stimulate DCs maturation. Furthermore, a fluorescent analogue of Sulfavants was synthesised for bioimaging studies in order to get information about the mechanisms responsible of immunogenic reaction. In particular, a derivative of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dye (BODIPY) was introduced on the hydrophobic moiety of Sulfavant R by new synthetic strategy. Moreover, higher amount of Sulfavant A was prepared by an improved and scalable synthesis. Finally, according to my PhD project, during an internship at the University of British Columbia, a synthetic strategy for the preparation of 1-O-alkyl-2-O-(2’-O-β-D-glucopyranosil-β-D-xylopyranosil)-glycerol has been designed; the above glycolipid was the main component of a natural bioactive mixture isolated by Professor Andersen and coworkers, from the UBC (Vancouver-Canada).

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