Abstract Title: Advancements in Nanomedicine: A Study on Lipid Nanoparticle Reliability and Formulation Optimization

Background: Lipid nanoparticles (LNP) have emerged as vital tools for delivering nucleic acids like messenger RNA (mRNA) and small interfering RNA (siRNA), playing a key role in the development of COVID-19 vaccines and RNA interference (RNAi)-based therapies for diseases including cancer and cardiovascular disorders. Despite their potential, inquiries into the comparative formulation efficiencies of LNPs when encapsulating siRNA versus mRNA payloads remain limited. Noteworthy challenges endure in optimizing LNP formulations, ensuring stability, scalability, and the mitigation of potential side effects. This study explores the encapsulation efficiency of LNPs for siRNA and mRNA, aiming to enhance the formulation of vaccines and gene therapies. We hypothesize that there is a difference in formulation efficiency and stability between siRNA and mRNA. Specific Aims: Aim 1: Compare the difference in formulation efficiency between siRNA and mRNA. Aim 2: Assess the stability of mRNA-LNP at room temperature.

Methods: Using siRNA targeting Firefly Luciferase and mRNA for NanoLuciferase, we prepared lipid formulations with ALC-0315, DSPC, cholesterol, and PEG-DMG. Following dialysis against PBS, we assessed cholesterol content, siRNA/mRNA concentration, and encapsulation efficiency through RiboGreen assays and determined particle size and polydispersity index (PDI) to evaluate stability. Results: In our experimental investigations, we have demonstrated the encapsulation formulation efficiencies for various components. Specifically, the encapsulation formulation efficiency for siRNA targeting Luciferase was found to be 85-95% (N=8, P<0.05), while that for mRNA encoding NanoLuciferase yielded a value of 80-90% (N=7, P<0.05). Furthermore, a comparative examination between siRNA and mRNA revealed distinct data regarding encapsulation formulation efficiency. Our findings also encompassed particle size measurements, indicating that siRNA-LNP had longer stability at room temperature compared to mRNA-LNP. mRNA-LNP had a significant increase in particle size compared to siRNA-LNP. The results pertaining to PDI showed that mRNA-LNP had a significant increase in PDI over a 12-hour time period.

Conclusion: These results provide valuable insights into the encapsulation properties and stability profiles of various components within lipid nanoparticles, contributing to the advancement of nanoparticle-based drug delivery systems. To further expand on these findings and advance research and therapeutic applications, future investigations may encompass evaluating encapsulation efficiency with plasmid DNA, exploring novel ionizable cationic lipids, and extending studies to encompass larger animal models.