Gene therapy refers to the use of DNA and RNA structured drugs with different molecular weight (oligo- and poly-nucleotides), structure (single or double strand), and action mechanism, for prevention or treatment of a variety of human diseases. The delivery of these molecules remains the largest issue for the widespread use of nucleic acid therapeutics. Indeed, nucleic acids suffer from biological instability and low target access. They can be degraded by nucleases and they result immunogenic. Their high molecular weight and negative charges prevent their absorption through biologic membranes (which are mostly negatively charged). Thereby, these bioactives are not able to be effectively internalized into cells and escape from endosomes to reach the cytosol or the nucleus. The lack of selective delivery by cell targeting also contributes to the failure of translation into medicine for most of therapeutic nucleic acids. So far, numerous studies have been devoted to realizing an effective nucleic acid delivery. However, the deficiency of biocompatible, biodegradable, non-immunogenic, highly efficient nucleic acid nanocarriers is still a crucial shortcoming to address. This thesis project aims at synthesizing and studying a “head-tail” oligosaccharide-based polycationic delivery system for nucleic acids. The macromolecular system presents a “star-like head” constituted by maltotriose, a biocompatible, biodegradable and non-immunogenic trisaccharide, containing numerous functionalizable hydroxyl-groups. Cationic moieties were conjugated to the “maltotriosyl-head”, providing the nucleic acid complexation through electrostatic interactions. Three types of cationic functions have been conjugated (guanidinium groups, tertiary amines or imidazole groups) to create different versions of the nanocarrier, aiming to optimize the nucleic acid complexation efficiency and making available different nucleic acid delivery profiles to expand the potential nanocarrier applications. A hydrophilic “tail” of polyethylene glycol (PEG) was bound to confer dispersibility and stealth properties to the nanocarrier. The project included two major parts: the nanocarrier synthesis and its physicochemical and biopharmaceutical characterization. Three major synthesis methods and several reaction conditions were investigated to set up an efficient multiple step synthesis procedure. A huge effort was dedicated to the optimization of each step purification, to the minimization of collateral subproducts and to the maximization of yields and reproducibility. Reliable analytical characterizations of both the intermediate and final products were set-up. In the first synthesis approach, the maltotriose hydroxylic group functionalization with cationic moieties (presenting the guanidinium group) was effectively realized before the final unsuccessful PEGylation. In the second attempted method, the PEGylation was carried out after the maltotriose hydroxylic group “activation” by esterification with acrylic anhydride (required for the cationic moieties subsequent conjugation by Michael addition), but a collateral PEGylation occurred. In the third synthesis method, the PEGylation was successfully performed before the maltotriose hydroxylic group esterification with acrylic anhydride, also allowing for the upcoming synthesis products purification by dialysis, optimizing the process. This last method resulted the best-performing one, even if further investigations are still required to realize an effective final Michael addition for the cationic moieties conjugation, avoiding interfering subproducts. Preliminary nucleic acid complexation studies performed by isothermal titration calorimetry and gel electrophoresis on the non-PEGylated guanidyl-functionalized macromolecular system suggested a PEG role in the polyplexes stabilization, but additional studies are required to verify this conclusion.

Supramolecular "head-tail" bioconjugates for nucleic acid delivery

CASAGRANDE, LISA
2021/2022

Abstract

Gene therapy refers to the use of DNA and RNA structured drugs with different molecular weight (oligo- and poly-nucleotides), structure (single or double strand), and action mechanism, for prevention or treatment of a variety of human diseases. The delivery of these molecules remains the largest issue for the widespread use of nucleic acid therapeutics. Indeed, nucleic acids suffer from biological instability and low target access. They can be degraded by nucleases and they result immunogenic. Their high molecular weight and negative charges prevent their absorption through biologic membranes (which are mostly negatively charged). Thereby, these bioactives are not able to be effectively internalized into cells and escape from endosomes to reach the cytosol or the nucleus. The lack of selective delivery by cell targeting also contributes to the failure of translation into medicine for most of therapeutic nucleic acids. So far, numerous studies have been devoted to realizing an effective nucleic acid delivery. However, the deficiency of biocompatible, biodegradable, non-immunogenic, highly efficient nucleic acid nanocarriers is still a crucial shortcoming to address. This thesis project aims at synthesizing and studying a “head-tail” oligosaccharide-based polycationic delivery system for nucleic acids. The macromolecular system presents a “star-like head” constituted by maltotriose, a biocompatible, biodegradable and non-immunogenic trisaccharide, containing numerous functionalizable hydroxyl-groups. Cationic moieties were conjugated to the “maltotriosyl-head”, providing the nucleic acid complexation through electrostatic interactions. Three types of cationic functions have been conjugated (guanidinium groups, tertiary amines or imidazole groups) to create different versions of the nanocarrier, aiming to optimize the nucleic acid complexation efficiency and making available different nucleic acid delivery profiles to expand the potential nanocarrier applications. A hydrophilic “tail” of polyethylene glycol (PEG) was bound to confer dispersibility and stealth properties to the nanocarrier. The project included two major parts: the nanocarrier synthesis and its physicochemical and biopharmaceutical characterization. Three major synthesis methods and several reaction conditions were investigated to set up an efficient multiple step synthesis procedure. A huge effort was dedicated to the optimization of each step purification, to the minimization of collateral subproducts and to the maximization of yields and reproducibility. Reliable analytical characterizations of both the intermediate and final products were set-up. In the first synthesis approach, the maltotriose hydroxylic group functionalization with cationic moieties (presenting the guanidinium group) was effectively realized before the final unsuccessful PEGylation. In the second attempted method, the PEGylation was carried out after the maltotriose hydroxylic group “activation” by esterification with acrylic anhydride (required for the cationic moieties subsequent conjugation by Michael addition), but a collateral PEGylation occurred. In the third synthesis method, the PEGylation was successfully performed before the maltotriose hydroxylic group esterification with acrylic anhydride, also allowing for the upcoming synthesis products purification by dialysis, optimizing the process. This last method resulted the best-performing one, even if further investigations are still required to realize an effective final Michael addition for the cationic moieties conjugation, avoiding interfering subproducts. Preliminary nucleic acid complexation studies performed by isothermal titration calorimetry and gel electrophoresis on the non-PEGylated guanidyl-functionalized macromolecular system suggested a PEG role in the polyplexes stabilization, but additional studies are required to verify this conclusion.
2021
Supramolecular "head-tail" bioconjugates for nucleic acid delivery
drug delivery
nucleic acids
cationic carriers
polyplexes
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12608/34892