Synthesis, Characterization, and Biomedical Applications of Polysaccharide-Modified mRNA-Lipid Nanoparticles

Abstract

Messenger RNA (mRNA) therapeutics have emerged as a transformative platform for vaccination, protein replacement therapy, gene editing, and cancer immunotherapy. Despite their rapid clinical advancement, broader implementation of mRNA technologies remains limited by challenges associated with nanoparticle manufacturing, delivery efficiency, repeated administration, and immune compatibility. The overall goal of this dissertation was to develop next-generation lipid nanoparticle (LNP) systems that address these challenges through innovations in both manufacturing technology and nanoparticle surface engineering. First, a low-cost automated fluidic synthesis platform was developed by repurposing a commercially available Ender-3 three-dimensional printer into a programmable nanoparticle manufacturing device. The resulting system enabled controlled and reproducible synthesis of nucleic acid-loaded LNPs while substantially reducing equipment costs compared with conventional commercial microfluidic platforms. Optimization of flow rate ratio (FRR) and total flow rate (TFR) parameters produced nanoparticles with favorable hydrodynamic diameters, low polydispersity indices, and high nucleic acid encapsulation efficiencies. These findings demonstrate the feasibility of decentralized and economically accessible nanomedicine manufacturing. Building upon this manufacturing platform, a modular post-formulation surface engineering strategy was developed to generate heparosan-functionalized lipid nanoparticles (HEP-LNPs). Heparosan, a naturally occurring polysaccharide and biosynthetic precursor of heparan sulfate and heparin, was covalently conjugated onto preformed LNPs through thiol-disulfide exchange chemistry. This approach enabled independent optimization of nanoparticle formulation and surface architecture while preserving nanoparticle integrity and mRNA encapsulation efficiency. Physicochemical characterization demonstrated that HEP-LNPs maintained colloidal stability, favorable particle size distributions, and high encapsulation efficiencies comparable to conventional PEGylated formulations. Systematic biological evaluation revealed that HEP surface engineering significantly enhanced mRNA delivery in professional antigen-presenting cells (APCs), including macrophages and dendritic cells. Screening studies identified SM-102/HEP-LNPs as the optimal formulation among the clinically relevant ionizable lipids evaluated. Notably, enhanced mRNA expression was not associated with increased nanoparticle uptake. Instead, live-cell trafficking studies demonstrated that HEP-LNPs exhibited reduced localization within Rab7-positive late endosomes and LAMP1-positive lysosomes while maintaining efficient progression through early endosomal compartments. These findings suggest that heparosan surface engineering improves functional mRNA delivery by reducing endolysosomal retention and promoting productive cytoplasmic release of mRNA cargo. The translational potential of HEP-LNPs was further evaluated through repeated-administration studies and SARS-CoV-2 spike S-2P mRNA vaccination models. HEP-LNPs maintained more sustained protein expression profiles following repeated subcutaneous administration and generated enhanced antigen-specific antibody responses compared with PEGylated controls. Importantly, these improvements were achieved without evidence of increased toxicity, pathological tissue remodeling, immune dysregulation, or detectable anti-heparosan antibody responses. Histopathological evaluation and immune cell profiling confirmed a favorable safety profile following repeated administration. Collectively, this dissertation demonstrates that advances in both nanoparticle manufacturing and biomimetic surface engineering can substantially improve the accessibility, efficacy, and safety of mRNA delivery systems. The low-cost automated synthesis platform developed herein provides a foundation for decentralized nanomedicine production, while heparosan-based surface engineering offers a promising PEG-free strategy for enhancing APC-targeted mRNA delivery. These findings establish intracellular trafficking as a critical determinant of delivery efficacy and provide a mechanistic and translational framework for the development of next-generation lipid nanoparticle therapeutics.