Carbohydrate-conjugated nanostructured platforms for selective interaction with enzymes and targeted delivery

Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by deficient or misfolded lysosomal enzymes, leading to substrate accumulation and cellular dysfunction. A current therapeutic approach involves pharmacological chaperones (PCs), molecules that stabilize misfolded enzymes and restore their activity. In this context, iminosugars (IMSs, carbohydrate mimetics in which the endocyclic oxygen is replaced by a nitrogen atom) are promising PCs for β-glucocerebrosidase (GCase)ii and N-acetylgalactosamine-6-sulfatase (GALNS), the defective enzymes in the LSDs Gaucher disease (GD) and Morquio A syndrome, respectively. The multimeric nature of these enzymes makes them suitable targets for multivalent ligand design. Indeed, recent studies have shown that multivalent IMSs enhance the stabilization of misfolded GCase and GALNS. However, PC therapy has not yet reached the market for GD, and further studies are required to generate more potent GCase stabilizers, including the development of new multivalent IMS systems and the elucidation of their intracellular fate in target cells, in order to further optimize the physicochemical properties of potential PC candidates. To address these needs, this PhD Thesis proposes the multimerization of selected IMSs on fluorescent quantum dots (QDs) and dendrimers, and investigates the ability of these newly developed multivalent IMS systems to modulate GCase activity and enable intracellular tracking. An analogous strategy was applied to the enzyme GALNS by replacing the IMS units in the dendrimers with the simple monosaccharide galactose. This design aimed to mimic the multivalent presentation of the natural GALNS substrate, keratan sulfate, whose repeating unit contains galactose derivatives. Furthermore, given recent evidence that GCase overexpression may be associated with chemotherapy resistance, mono- and multivalent IMSs were also evaluated for the first time as adjuvant agents in combination with a conventional anticancer drug (cisplatin), in order to assess potential synergistic effects and enhance therapeutic efficacy. Another current therapeutic approach for LSDs is enzyme replacement therapy (ERT). However, its efficacy is limited by enzyme instability and poor tissue penetration. To overcome these limitations, supramolecular poly(allylamine)/dextran glyco-nanoparticles (PAH:DEX NPs) were developed for the delivery of the enzyme iduronate-2-sulfatase (IDS), whose deficiency leads to LSD Hunter syndrome. The aim of this part of the PhD work was to encapsulate, and thus protect, IDS inside PAH:DEX NPs and then enabling its pH-responsive release. The DEX component further contributes to facilitating cellular uptake and efficient intracellular delivery. Overall, this PhD Thesis focuses on the design and development of carbohydrate-based nanoplatforms for therapeutic applications. Multivalent arrangements of sugars and sugar mimetics have been engineered on dendrimers and fluorescent nanoparticles (quantum dots) to modulate the activity of carbohydrate-processing proteins, with a particular focus on enzymes implicated in LSDs and cancer. Beyond selective targeting, some nanoplatforms were developed as tuneable, stimuli-responsive systems capable of responding to pH changes, while others exploited their inherent physicochemical properties to perform additional functions, such as intracellular tracking. Thus, this PhD work further illustrates how glyconanotechnology can provide integrated strategies for targeted delivery and modulation of biological targets, thereby laying the foundation for the development of next-generation carbohydrate-based therapeutic nanosystems.