DESIGN AND EVALUATION OF POLYMER-LIPID CONJUGATES FOR mRNA DELIVERY SYSTEMS

Lipid nanoparticles (LNPs) have emerged as the most advanced and clinically validated delivery systems for ribonucleic acid (RNA), as demonstrated by their success in mRNA vaccines and expanding therapeutic applications. Their clinical translation, however, depends not only on efficient RNA encapsulation and delivery but also on the ability to preserve colloidal stability during storage and circulation. Among their components, poly(ethylene glycol) (PEG)-lipid conjugates play a pivotal role by forming a protective hydrophilic corona, suppressing aggregation, enhancing shelf-life, and prolonging systemic circulation. Despite these advantages, however, PEGylation is not without drawbacks. Particularly. PEGylation might reduce cellular uptake, induce anti-PEG immune response and not provide enough long-term stability. These issues highlight the need for next-generation stabilizers that maintain or improve PEG performance while overcoming its intrinsic disadvantages. This thesis systematically investigates PEG alternatives with the goal of defining design principles for polymers that can stabilize RNA-loaded LNPs without compromising delivery efficiency or safety. The central objectives are: (i) to identify and characterize polymers able to replicate or surpass the steric stabilization of PEG-lipids, (ii) to evaluate their impact on particle size, dispersity, encapsulation efficiency, and storage stability, (iii) to assess their performance under physiologically relevant conditions, and (iv) to establish their capacity for effective and biocompatible RNA delivery. The thesis is structured into four chapters. Chapter 1 provides a comprehensive overview of PEG-lipids and their limitations, with emphasis on immunogenicity, accelerated clearance, and delivery inefficiencies. It examines emerging polymer classes, including polysarcosines, poly(2-oxazoline)s, poly[oligo(ethylene glycol) methacrylate] (POEGMA), and polyzwitterions, highlighting their influence on colloidal stability, biocompatibility, and delivery performance. Chapter 2 explores the role of polymer topology in nanoparticle stabilization, with particular focus on cyclic polymers. Their synthesis, antifouling properties, and ability to improve colloidal stability are discussed as promising alternatives to conventional linear topology. Chapter 3 investigates polyzwitterion-lipid conjugates based on poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). Both single- and double-tail PMPC-lipids are evaluated in mRNA-LNP formulations, with detailed in vitro and in vivo studies on transfection efficiency, cytotoxicity, protein expression, and storage stability compared to PEG benchmarks. Finally, Chapter 4 examines cyclic PEG (cPEG) as a topological alternative to linear PEG. The impact of cPEG-lipid conjugates on LNP physicochemical characteristics, encapsulation efficiency, and colloidal stability is assessed, together with transfection outcomes in vitro and biological performance in vivo. Overall, this work highlights how both chemical nature and polymer topology dictate the performance of stabilizing lipids in LNPs. By integrating systematic design, synthesis, and biological evaluation, it contributes to the rational development of new components for RNA nanomedicine, offering strategies to overcome the PEG dilemma and advance the next generation of LNP platforms.