SYNTHESIS AND OPTIMIZATION OF DNA NANOPARTICLES FOR THERAPEUTIC APPLICATIONS

Abstract

Nanoparticles (NPs) represent a versatile platform for therapeutic delivery and diagnostic applications due to their nanoscale size, enhanced surface-to-volume ratio, and capacity for targeted delivery. Among various types of NPs, chitosan nanoparticles (CsNPs) have gained significant attention due to their biodegradability, biocompatibility, and mucoadhesive properties. Despite these advantages, CsNPs face challenges such as instability during storage and variable transfection efficiency. This thesis addresses these limitations by optimizing the synthesis, storage, and functional properties of CsNPs, specifically for DNA delivery and non-viral vaccine applications. Through lyophilization and sonication, CsNPs retained their transfection efficiency and stability over long-term storage, demonstrating nearly identical transgene expression levels to freshly prepared particles after three months at 4 °C. Optimal lyophilization conditions, including a duration of 24 hours and a batch volume of 3000 µL, yielded the highest transfection efficiency, emphasizing the importance of process parameters in nanoparticle functionality. Sonication post-lyophilization further enhanced DNA expression by reducing aggregation and maintaining spherical morphology. The N:P ratio significantly influenced CsNP stability and transfection efficiency, with ratios of 2:1 and 3:1 offering optimal DNA condensation, cellular uptake, and biocompatibility. These findings align with previous studies suggesting moderate N:P ratios are ideal for gene delivery applications. Additionally, this study investigates the physicochemical characteristics, cellular uptake dynamics, and cell cycle-dependent uptake of CsNPs to optimize their potential as delivery systems. The combination of fluorescent dyes (FITC and Cy5) significantly altered the surface charge of CsNPs, reducing their cellular uptake efficiency, with double-labeled nanoparticles showing the lowest uptake. Time-dependent uptake analyses revealed that CsNPs localized near the cell membrane within 2 hours post-treatment, internalized into the perinuclear region by 12 hours, and plateaued at 24 hours, with FITC remaining detectable for up to 48 hours. Cell cycle synchronization studies showed higher CsNP uptake in G2/M phase cells compared to S and G0/G1 phases, likely due to increased membrane permeability and nuclear accessibility. Additionally, lower concentrations of synchronization agents (200 nM) enhanced transfection efficiency, highlighting the role of reduced cytotoxicity. These findings underscore the importance of dye-labeling optimization, intracellular dynamics, and cell cycle synchronization in designing efficient nanoparticle-based delivery systems. The study also explored CsNPs encoding the SARS-CoV-2 Spike glycoprotein (S-CsNPs) as immunogenic agents, administered via oral (PO) and intramuscular (IM) routes in combination with the CpG 7909 adjuvant. IM delivery elicited a robust humoral immune response, while PO delivery modulated immune tolerance. Co-administration of CpG 7909 significantly enhanced IgG antibody levels for both routes, demonstrating its potential as a versatile immunostimulatory agent. The findings obtained suggest that S-CsNPs, particularly when paired with CpG, hold promise for mucosal and systemic immune-stimulator applications, offering advantages in ease of administration and scalability. Despite these advancements, limitations remain, including the need for long-term studies to assess the durability of the immune response and further evaluation of S-CsNPs as immune-stimulator vehicle. This work provides a foundation for future research to optimize vaccine formulations and delivery strategies, advancing CsNP applications in gene delivery and immunization, particularly in scenarios requiring extended storage and rapid deployment.