Advanced structural and morphological characterization by NMR technologies of different biomolecules and biotechnological systems

This research work focuses on the advanced characterization of pharmaceutical systems which are used as delivery platforms for nucleic acids and proteins, using advanced Nuclear Magnetic Resonance (NMR) techniques. The delivery systems studied in this doctoral work are liposomes, Lipid Nanoparticles (LNPs) and Red Blood Cells (RBC). There is indeed an urgent need to enhance understanding of structural attributes that can be linked to functional variations, thereby guiding rational formulation design. Comprehensive characterization to increase product understanding of these systems is challenging due to their complexity (size, composition, dynamics). Additionally, the stability of these systems is often a concern, requiring thorough analytical investigations overtime and across different batches to ensure quality and consistency. NMR presents distinct advantages for investigating delivery systems by integrating several unique capabilities. It provides atomic resolution that enables a detailed examination of individual components, within a complex architecture. It’s a non-destructive technique allowing for the analysis of the encapsulated cargo in intact complexes. There is a wide set of experiments to gain insights into dynamics of systems, correlating structural attributes with activity. Furthermore, NMR allows to monitor stability over time under various storage and stress conditions, enabling the elucidation of degradation pathways. These comprehensive features make NMR an invaluable tool for advancing the design and optimization of delivery systems in pharmaceutical applications, ensuring their efficacy and reliability. The first study of this doctoral work was focused on lipid quantification in liposomes. To optimize formulation and support large-scale production, precise and rapid analytical methods are necessary to monitor lipid content during formulation and follow degradation processes overtime. Two NMR methods were refined and applied for this scope: a conventional method employing an internal standard and the PULCON NMR method, in which the reference is external for streamlined reproducibility and accelerated processing, particularly beneficial for industrial-scale applications. Through comparison to Ultra-Performance Liquid Chromatography coupled with Evaporative Light Scattering Detection (UPLC-ELSD), this study demonstrates NMR as a viable and efficient alternative for lipid quantification, with rapid data acquisition and the need of only one reference standard. Furthermore, we worked on the characterization of proteins encapsulated in Red Blood Cells, which are gaining interest as smart delivery systems, ensuring biocompatibility and long drug circulation. The research provided, utilizing NMR techniques, an in-depth structural analysis of the higher-order structure (HOS) of proteins encapsulated within erythrocytes, which is essential for maintaining biological function and therapeutic effectiveness. A semi-quantitative analysis of the encapsulated enzymes was also performed using NMR, providing a tool for fast quantification which could guide optimization of the encapsulation protocol, applicable also to other cargos. Finally, lipid nanoparticles (LNPs) were investigated, particularly focusing following the PEG shedding phenomenon using Pulsed Gradient Spin Echo (PGSE) NMR. PEG shedding rate affects the biodistribution of LNPs and should be considered carefully when designing the formulations for each specific application. Moreover, concern regarding the stability of PEG before injection is growing, making it necessary to develop a method as quality control and to guide a rational design of particles. For this purpose, the analysis assessed the release of PEG under different storage and stress conditions. In addition, a prediction of PEG shedding rate in vivo was performed by adding Bovine Serum Albumin (BSA) to the LNP solutions, highlighting variability among different formulations that should be considered in the design of particles. Overall, this thesis contributes to the advancement of drug delivery system characterization by refining analytical methodologies to enhance quality controls of batches and to deepen our understanding of formulations. These findings will be instrumental for the rational design of delivery systems, ultimately improving their effectiveness. This PhD project originates from a collaboration between GSK (Siena, IT) and the Magnetic Resonance Center (CERM) of the University of Florence (Florence, IT).