Investigations into mRNA Lipid Nanoparticles Shelf-Life Stability under Nonfrozen Conditions

Abstract

mRNA LNPs can experience a decline in activity over short periods (ranging from weeks to months). As a result, they require frozen storage and transportation conditions to maintain their full functionality when utilized. Currently approved commercially available mRNA LNP vaccines also necessitate frozen storage and supply chain management. Overcoming this significant inconvenience in the future is crucial to reducing unnecessary costs and challenges associated with storage and transport. In this study, our objective was to illuminate the potential time frame for nonfrozen storage and transportation conditions of mRNA LNPs without compromising their activity. To achieve this goal, we conducted a stability assessment and an in vitro cell culture delivery study involving five mRNA LNPs. These LNPs were constructed by using a standard formulation similar to that employed in the three commercially available LNP formulations. Among these formulations, we selected five structurally diverse ionizable lipids�C12−200, CKK-E12, MC3, SM-102, and lipid 23�from the existing literature. We incorporated these lipids into a standard LNP formulation, keeping all other components identical. The LNPs, carrying mRNA payloads, were synthesized by using microfluidic mixing technology. We evaluated the shelf life stability of these LNPs over a span of 9 weeks at temperatures of 2−8, 25, and 40 °C, utilizing an array of analytical techniques. Our findings indicated minimal impact on the hydrodynamic diameter, zeta potential, encapsulation efficiency, and polydispersity of all LNPs across the various temperatures over the studied period. The RiboGreen assay analysis of LNPs showed consistent mRNA contents over several weeks at various nonfrozen storage temperatures, leading to the incorrect assumption of intact and functional LNPs. This misunderstanding was rectified by the significant differences observed in EGFP protein expression in an in vitro cell culture (using HEK293 cells) across the five LNPs. Specifically, only LNP 1 (C12−200) and LNP 4 (SM-102) exhibited high levels of EGFP expression at the start (T0), with over 90% of HEK293 cells transfected and mean fluorescence intensity (MFI) levels exceeding 1. Interestingly, LNP 1 (C12−200) maintained largely unchanged levels of in vitro activity over 11 weeks when stored at both 2−8 and 25 °C. In contrast, LNP 4 (SM-102) retained its functionality when stored at 2−8 °C over 11 weeks but experienced a gradual decline of in vitro activity when stored at room temperature over the same period. Importantly, we observed distinct LNP architectures for the five formulations through cryo-EM imaging. This highlights the necessity for a deeper comprehension of structure−activity relationships within these complex continued...

Pharmacokinetic Analyses of a Lipid Nanoparticle-Encapsulated mRNA-Encoded Antibody against Rift Valley Fever Virus

“…However, some evidence about absorption and biodistribution is needed to complete the pharmacokinetics profile, especially in the study of therapeutic antibodies . Tenth, mRNA-LNP was injected in vivo , and conditions such as body temperature, pH, and various enzymes would affect the LNP stability and biocompatibility. , In this paper, we only addressed some common key parameters on the stability (Figure ), and it would be expected that future evaluations might explore other parameters likely to be encountered following administration in animals. However, in this experiment, the main focus was on successfully expressing the antibody and establishing its pharmacokinetic analyses.…”

Lipid Nanoparticle (LNP) Delivery Carrier-Assisted Targeted Controlled Release mRNA Vaccines in Tumor Immunity

“…These vaccines work by delivering mRNA sequences encoding tumor-specific antigens, driving antigen expression within host cells and promoting immune system activation. Dendritic cells are key cells in the immune system that are able to take up exogenous antigens and present them to T cells to initiate specific immune responses [ 101 , 102 , 103 , 104 ]. mRNA vaccines activate dendritic cells to take up and present tumor-specific antigens, triggering the activation and proliferation of T cells [ 105 ].…”

Lipid Nanoparticle (LNP) Delivery Vector-Assisted Targeted Controlled Release mRNA Vaccines in Tumor Immunity

“…These vaccines work by delivering mRNA sequences encoding tumor-specific antigens, driving antigen expression within host cells, and promoting immune system activation. Dendritic cells are key cells in the immune system that are able to take up exogenous antigens and present them to T cells to initiate specific immune responses [101][102][103][104] . mRNA vaccines activate dendritic cells to take up and present tumor-specific antigens, triggering the activation and proliferation of T cells [105] .…”