Recombinant ApoE3 corona plus sucrose vitrification endows mRNA-LNPs with Freeze-Thaw and -80 °C stability

Lipid nanoparticles (LNPs) enable clinical mRNA delivery but remain paradoxically fragile: storage at -80 °C slows RNA hydrolysis yet triggers lipid phase separation and ice damage. Conventional sugar cryoprotectants immobilize water, but incompletely stabilize the lipid matrix, leaving ＞30 % activity loss after one month. Here we report a dual, chemically-defined strategy that (i) pre-adsorbs recombinant apolipoprotein E3 (ApoE3) to which may help stabilize lipid packing via reversible interfacial binding, and (ii) vitrifies the surrounding solution with 10 % (w/v) sucrose. Using two representative ALC-0315 backbones with divergent potencies, the dual strategy preserved > 90 % mRNA encapsulation, size uniformity and luciferase expression after 4 weeks at -80 °C and three freeze-thaw cycles. In C57BL/6 mice, liver bioluminescence rose ∼ 3-fold relative to fresh untreated LNPs without elevating interleukin-6(IL-6) or organ toxicity. Thawed samples retained > 70 % potency for 72 h at 4-25 °C, exceeding the ≤ 12 h handling window of authorized COVID-19 vaccines. Both excipients can be produced at cGMP scale and are added in a single step, facilitating integration into existing manufacturing and cold-chain workflows. By combining pre-adsorbed ApoE3 with sucrose vitrification, this strategy provides a practical route to more durable mRNA vaccines and gene therapies while avoiding lyophilization or plasma-derived additives.