Enhancing RNA Payload and Temperature Stability and Activity with Cationic Peptide-Coated Zinc Oxide Nanoparticles

The lipid nanoparticle (LNP) mRNA vaccine was first tested through clinic but suffered from relatively low RNA payloads and poor temperature stability. Our lab patented a protamine-coated particle approach for temperature-stabilizing DNA vaccines, translating this successfully to the clinic. In subsequent work, we have characterized RNA interaction and delivery by zinc oxide nanoparticles, filing a patent most recently entitled RNA-stabilizing nanoparticles, similarly utilizing protamine-coated zinc oxide nanoparticles for RNA. Here, we present this data for the first time. Briefly, ZnO, ZnO-protamine, and ZnO-protamine-RNA were characterized by size and zeta potential analyses and the RNA-loaded nanoparticles were visualized by transmission electron microscopy. UV spectroscopic analysis demonstrated up to 95-98% loading efficiency with protamine and approximately 75% loading efficiency with LL37, another cationic antiviral peptide. Elution of the RNA isolated from the particles afforded a calculation in three independent trials where RNA payloads ranged from 18 to 45 μg of RNA per 0.5 mg of coated particles. Circular dichroism (CD) analysis indicated that binding of RNA to ZnO NPs stabilized, enhancing the pattern with a clear dependence on the RNA:ZnO stoichiometry. Enhanced temperature stability was shown by differential scanning calorimetry (DSC), gel electrophoresis, and in vitro mRNA expression analysis. Using poly I:C RNA with a well-defined melting point (64.3 ± 0.32 °C), formation of the ZnO:RNA complex increased the RNA melting point (70.9 ± 0.62 °C). After refrigerated or room-temperature storage or incubation at 30, 40, or 50 °C, RNA comigration with the control RNA was recovered from all samples, exposed to either 14 or 100 nm ZnO, and coated with protamine. Furthermore, the ZnO-protamine-mRNA samples retained significantly higher expression activity when incubated at these elevated temperatures. Finally, the ZnO-protamine-mRNA was functionally active for in vitro translation, in cell extracts, and in cells for expression of GFP, luciferase, and COVID spike protein. These data support further preclinical development of ZnO-protamine-mRNA.