THE PERMEATION OF BIOMACROMOLECULES THROUGH HUMAN SKIN: AN INSIGHT INTO THE FAMILY OF GLYCOSAMINOGLYCANS

This Ph.D was aimed to elucidate the physicochemical parameters that rule the penetration of different classes of glycosaminoglycans (GAGs) through human skin, with particular focus on hyaluronan (HA) and heparins. The research activity involved: 1) the investigation of the influence of the main structural features (sequence, molecular weight (Mw), polarity, and conformational behavior) on the percutaneous absorption of HA, heparin (UFH) and low molecular weight heparins (LMWHs); 2) the fine-tuning of a suitable enhancement strategy to improve the permeation of UFH through human epidermis. The overall results confirmed that Mw represents a fundamental factor in determining the permeability properties also of such polysaccharides. Indeed, in in vitro studies performed by using HA having different chain lengths, the diffusion through the skin decreased increasing the polymer’s molecular mass. Similarly, LMWHs sodium salts permeated to a larger extent with respect to UFH and their permeated amounts varied with Mw according to an exponential relationship. However, the key parameter driving the permeation of such macromolecules through the skin resulted to be the polymer flexibility. In fact, the introduction of sulfate groups on HA chains, causing the disruption of the helical motif, led to less ordered structures, which were able to cross the skin to higher extent compared to unsubstituted HA, despite their increased polarity. Accordingly, the presence of divalent cations in LMWHs solution, limiting the chain flexibility through the formation of intramolecular chelates, negatively affected the skin permeability properties of calcium nadroparin. Furthermore, both classes of polysaccharides in study demonstrated to interact with stratum corneum components, in particular keratins, as confirmed by confocal miscroscopy images (HA) and ATR-FTIR spectra (UFH). Since the physicochemical features of these polysaccharides (hydrophilicity, negative charge and high Mw) precluded a significant diffusion through the human epidermis, the phage display technique was exploited to screen a skin penetrating peptide which could enhance the skin permeation of such molecules, allowing their transdermal delivery. The in vitro screening allowed to identify a neutral peptide, DRTTLTN, which resulted to be able to increase the UFH flux of 34-fold after chemical conjugation. These results demonstrated the suitability of skin penetrating peptides to improve the skin delivery of polysaccharide macromolecules.