Polyaminosquaramides (PASQUA) as gene delivery vehicles and cell labelling agents

eng The use of squaramides in the field of biological and supramolecular chemistry includes significant applications such as powerful anion transporters or fluorescent probes. Their widespread use is primarily due to their synthetic accessibility and their ability to establish intermolecular interactions, particularly hydrogen bonds. However, the incorporation of squaramides in polymers is not as common, and the application of polysquaramides in biological chemistry has not been described to date. The use of cationic polymers in the field of gene delivery as substitutes for viral vectors is gaining importance due to advantages such as greater scalability, the capability to transport nucleic acids without size limitations, and the absence of immune responses. In this context, the ability of squaramides to establish intermolecular interactions with anions, combined with their potential to synthesise polymers, makes them ideal candidates for the development of polysquaramides with applications in nucleic-acid delivery. This thesis, titled “Polyaminosquaramides (PASQUA) as Gene Delivery Vehicles and Cell Labelling Agents”, explores the synthesis and characterisation of a family of polysquaramides, as well as their subsequent evaluation as gene delivery vehicles. This along with its antecedents from the doctoral thesis of PhD Francisca Orvay, represents the first example of polysquaramides applied to nucleic-acid transport and to the field of biological chemistry in general. The thesis is divided into eight chapters, with the synthesised polymers grouped into subfamilies based on their structural similarities. The first chapter introduces fundamental concepts of polymer chemistry, gene therapy, and nucleic acid delivery, highlighting the applicability of certain polymers in these fields. It also provides details on the structures and key advantages of the main polymers currently used for nucleic acid delivery. Additionally, the chapter introduces the "squaramide" unit, discussing its applications across various fields and its role in polymer formation. Finally, it reviews the key contributions of the research group to the study of squaramides, with a special focus on the results obtained by Francisca Orvay in her doctoral thesis, entitled “Synthesis and characterisation of polyaminosquaramides: aggregation studies and biological activity” This earlier work introduced PASQUA polymer (polyaminosquaramide) for the first time. The second chapter outlines the main objectives of this doctoral thesis, which are developed in detail between Chapters Three and Six. Each of these chapters includes its motivation and objectives, the experimental results and discussion, conclusions, and the corresponding experimental section. Chapter Three describes the optimisation process for the synthesis and purification of PASQUA, resulting in a new material with enhanced performance, designated as PASQUA+. Additionally, it includes the validation of the material evaluating its interaction with nucleic acids and its capability as a transfection agent for transporting plasmids of different sizes and morphologies. Chapter Four focuses on the design of various polyoxyethyleneaminosquaramides and their evaluation as transfection agents to study how reducing the number of ionisable nitrogen atoms in the repeating unit affects transfection efficiency. Chapter Five describes the synthesis of new polyaminosquaramides modified with primary amino groups throughout their structure. This strategy increases the number of ionisable nitrogen atoms in the repeating unit and offers the possibility of functionalising the resulting polymers through post-polymerisation reactions. As with the previous chapters, the biological evaluation of the synthesised polymers is also included. Chapter Six centres on the synthesis of fluorescent polyaminosquaramides through the conjugation of fluorophores to the modified polyaminosquaramides with primary amino groups described in Chapter Five. Alongside their synthesis and characterisation, this chapter explores their application as transfection agents and cell-labelling tools. Finally, Chapters Seven and Eight compile the general conclusions and bibliography, respectively.