Phd in MORPHOGENESIS AND TISSUE ENGINEERING

Thesis title: Development of DNA vaccine encapsulated in multicomponent lipid nanoparticles.

Rationale: Lipid nanoparticles (LNPs) have emerged as significant players in nanomedicine, serving as carriers for therapeutic agents such as anticancer drugs, short interfering RNA (siRNA), and more recently, messenger RNA (mRNA) in the context of COVID-19 vaccine development. However, the successful encapsulation and delivery of plasmid DNA (pDNA) within LNPs remain challenging due to technological limitations and gaps in our understanding of their structural and functional properties. General Objectives: To overcome these challenges, we sought to investigate the interrelation between tuning the manufacturing and microfluidic parameters with the physicochemical properties and efficacy of LNPs, aiming to gain valuable insights into the essential characteristics required for an effective DNA-encapsulating LNP. Armed with this knowledge, our research endeavors encompassed the development, characterization, and evaluation of a library comprising 17 multicomponent DNA-loaded LNPs. From this library, we aimed to select the formulations exhibiting proper attributes for a possible in vivo application. These chosen formulations underwent rigorous assessment to determine their transfection efficiency, toxicity profiles, and nanostructure in vitro. Subsequently, the most promising formulation was selected for in vivo delivery of a DNA vaccine. To enhance its versatility, we further optimized this formulation using biomolecules, enabling its potential application in diverse contexts such as gene therapy. Experimental Design and Methods: The multicomponent DNA-loaded LNPs were synthesized using microfluidic mixing techniques with the aid of an automated device that allowed fine-tuning of various parameters. Subsequently, all 17 LNPs were subjected to size and surface charge characterization through Dynamic light scattering and microelectrophoresis. Moreover, the eight selected formulations underwent comprehensive in vitro investigations to assess their capability in delivering DNA and to evaluate potential toxic effects. Transfection experiments and cell viability assays were performed to achieve this evaluation. Moreover, we delved into the nanostructural properties of the selected LNPs using transmission electron microscopy and synchrotron small-angle X-ray scattering (SAXS). The most effective formulation, capable of encapsulating a DNA vaccine for HER2+ tumors, was subsequently employed in in vivo mice vaccination experiments and further optimized for gene therapy applications. Results: The manipulation of microfluidic and manufacturing parameters exerted a profound influence on the chemical-physical characteristics and nanostructure of the gene delivery system generated. Consequently, these factors significantly impacted the biocompatibility and efficacy of the system. Among the various formulations, we carefully selected the best performing one, which demonstrated remarkable potential as a vaccine nanocarrier. This successful demonstration paved the way for its practical application in this context. Furthermore, to enhance its functionality, we endowed the LNP with a biomolecular coating, specifically pDNA, which not only improved its efficiency but also altered its surface features, enabling the LNP to evade recognition by the immune system. This capability opens up exciting possibilities for gene therapy applications. Conclusions: Through rigorous evaluations, we successfully identified a highly promising LNP formulation capable of serving as a potential vaccine candidate for combating HER2+ tumors. This formulation, suitable for DNA vaccine delivery can be further optimized using DNA gaining good potential for diverse in vivo applications. Our findings significantly contribute to advancing the understanding and knowledge of LNP-based delivery systems, which in turn can have far-reaching implications for the advancement of vaccination and gene therapy strategies.