Advanced Drug Delivery Nanosystems

The basis of my thesis work is the synthesis and characterization of innovative tools relevant to the pharmaceutical and technological fields. My attention is focused on the development of original methodologies aimed at improving the pharmacokinetic and therapeutic profile of drugs through a multidisciplinary approach. In particular, the third chapter of my thesis work deals with the development of an innovative strategy for the covalent selective proteins PEGylation by exploiting the N-acetylhexahistidine (Ac-His-6) as a model of poly-histidine tag. The lack of selectivity and site-specificity of the classic PEGylation strategies led us to study the reactivity of the Morita-Bayles-Hillman adducts (MBHA) as new functional tool to activate PEG chains for subsequent coupling reactions. These molecules, thanks to a concerted addition-elimination mechanism, react with imidazole ring without catalyst and in the presence of water. Therefore, a small series of MBHA acetates bearing a clickable propargyl group were synthesized and reacted with imidazole in order to study their reactivity Unexpectedly, the synthesized MBHA acetates react with the imidazole ring forming a biadduct when the reaction is carried out in acetonitrile /phosphate buffer (PBS), whose presence was also confirmed in the reaction with N-acetylhistidine. Subsequently, after insertion of PEG chain on MBHA scaffold by means of “click” reaction, the reactivity of MBHA in PBS was exploited with Ac-His-6. The polymeric materials obtained were characterized by mass spectrometry, NMR spectroscopy and photophysical studies, confirming the presence of considerable amounts of biadduct residues in the polymeric backbone. The macromolecular mechanism leading to the multi-functionalization of Ac-His-6 has been extensively studied. Given the amphiphilic nature of PEG-MBHA derivatives, they self-aggregate in water, generating central shell structures that act as nanoreactors responsible for the multiple attacks on the Ac-His-6 tag. Finally, this peculiar reactivity was explored by reacting water soluble MBHA with an antibody fragment containing histidine residues only in the tag. In the fourth chapter the main features and the peculiar polymerization mechanism of 3-phenylbenzofulvene monomers are reported. They are subjected to spontaneous polymerization by simple removal of the solvent and showed thermoreversible polymerization/depolymerization features a high predisposition to molecular manipulation and tended to generate nanostructured macromolecular aggregates. The polymerization mechanism has been extensively studied through structure-activity relationship studies of the prototype monomer BF-1 and it was named "affinity polymerization". Benzofulvene derivatives have a strong tendency to π‐stacking interactions, that have been considered responsable of spontaneous polymerization of monomeric units of BF1 and its congeners. Thanks to these interactions, benzofulvene monomers organize in columnar aggregates, which are compacted up to the critical distance of polymerization. The number of monomers and the location of the substituents affect the number of aggregated monomers needed to reach the critical polymerization distance. It has been discovered that the phenyl substituent in position 3 of the indene ring has a crucial role in the progress of the polymerization process since it establishes stacking interactions able to influence the aggregation phase between the monomers. Therefore, a small series of 3-phenyl benzofulvenic monomers substituted with an electron withdrawal group or electron donors were synthesized and characterized. The results of SEC-MALS, Single Crystal X-Ray Diffraction studies on 3-phenyl benzofulvene monomeric models and the comparison of the NMR spectra of polymers with those of monomer unit, confirmed the crucial role of the hydrogen in 2'-position in the spontaneous polymerization. Different strategies of molecular manipulation of the polybenzofulvene backbone for the insertion of PEG side chains in different positions are illustrated to improve the weak interaction with water and to reduce their strong tendency to aggregate, which limit their use in pharmaceutical and technological fields (nano-encapsulation and drug delivery). Therefore, a new family of PEGylated polymeric brushes was synthesized combining the cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) “click” reaction and the intriguing spontaneous thermoreversible polymerization mechanism by affinity, both in a grafting through (GT) and in a grafting on (GO) approach. Finally, the fifth chapter describes the development of a technological platform for the coating of surfaces of various nature with low molecular weight hyaluronic acid (HA) in order to obtain a biomimetic and therefore biocompatible coating (biomimetic biocompatible hyaluronic coating, BBHC). In this regard, the research group of Prof. Cappelli, has developed a synthetic procedure to transform HA into an HA-FA copolymer, by functionalization with ferulic acid, combining the exceptional properties of the two natural compounds. Subsequently, the grafting procedure was modified using a derivative of the ferulic acid functionalized with a propargyl group in order to introduce "clickable" propargyl groups into the hyaluronic acid chain, that is usable in subsequent "click chemistry" reactions such as the 1,3-dipolar azide-alkino cycloaddition catalyzed by copper (I) (CuAAC). This procedure was optimized using low molecular weight HA to give grafted copolymers (HA-FA-Pg) with a variable grafting degree in the range from 10% to 35%. In particular, the HA was anchored on the hydrophobic surface of solid lipid nanoparticles (SLN) and on the surface of magnetite nanoparticles by click reaction (CuAAC) between the azido-functionalized surfaces and the HA derivatives bearing propargylated ferulic groups.