Lipid Nanoparticles (LNPs) have recently been used in COVID-19 vaccines to protect and deliver mRNA to cells in the body to code for proteins which train the immune system to fight the virus. Without LNPs, mRNA would degrade in the blood stream and never make it inside of cells to do its job. The success of LNPs over the past few years at delivering genetic material to the body has been challenged with the inflammatory nature of LNPs. While immuno-stimulation is a plus point for a vaccine, it could be lethal to critically ill patients with pre-existing inflammation, such as those with acute respiratory distress syndrome, which can occur after COVID infection. Our lab had recently shown that lipid nanoparticles exacerbate pre-existing inflammation several fold in mouse models. Of the several lipids involved in making an LNP, the ionizable lipid component, which allows the LNP to degrade inside cellular lysosomes and release the mRNA, has been identified to cause inflammation through the toll-like receptor and NLRP3 pathway. This inflammation was able to be reduced by co-delivering dexamethasone, a clinically safe upstream inflammation inhibitor. However, dexamethasone as a soluble drug in the blood stream is not targeted to any specific organ, meaning that uptake to the organ of interest is inefficient. Since the LNPs themselves are targeted to the diseased organ by conjugating antibodies to their surface, it would be ideal to package dexamethasone in the LNP so that it can be delivered along with the mRNA cargo in the LNP.

This is challenging because dexamethasone is likely not lipophilic enough to incorporate into the organic phase of the LNP on its own, leading to unstable LNPs with low dexamethasone loading efficiency and high leakage. A solution is to add a long fatty acid chain, specifically a palmitate group, to the dexamethasone, leading to a very lipophilic precursor drug called dexamethasone palmitate, or DXP, with a greater chance of incorporating well into the LNPs. DXP is readily metabolized by esterase into the active dexamethasone in blood. In this project, we studied the efficacy of loading DXP into the organic phase of siRNA-LNPs. We used the Nanoassemblr Ignite platform to mix both the organic phase, containing the lipids and DXP, and the aqueous phase, containing the mRNA, under laminar flow. We use dynamic light scattering to quantify the homogeneity of the LNPs that were produced, achieving a mean diameter of 80 nm with a low polydispersity index. We also use a fluorescent assay to determine the percentage of mRNA that was encapsulated in the LNP and achieved an approximately 90% encapsulation efficiency. We then used ultra high-pressure liquid chromatography (UPLC) to determine the loading efficiency and leak of the DXP. The initial loading efficiency was 66%, and the total leak plateaued at approximately 36% after 6 days. Our robust LNPs are now being tested in vivo in models of inflammation.