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

Area of Science:

• Biotechnology
• Materials Science
• Nanotechnology

Background:

• Lipid nanoparticle (LNP) mRNA vaccines face challenges with low RNA payloads and poor temperature stability.
• Previous work successfully applied protamine-coated particles for temperature-stabilizing DNA vaccines.
• Zinc oxide nanoparticles (ZnO NPs) are explored for RNA interaction and delivery, with a focus on stabilization.

Purpose of the Study:

• To present data on protamine-coated zinc oxide nanoparticles for RNA stabilization and delivery.
• To characterize the physicochemical properties and RNA loading efficiency of these nanoparticles.
• To evaluate the enhanced temperature stability and in vitro functional activity of RNA encapsulated within ZnO-protamine nanoparticles.

Main Methods:

• Characterization of ZnO, ZnO-protamine, and ZnO-protamine-RNA nanoparticles using size, zeta potential, and transmission electron microscopy.
• UV spectroscopy for RNA loading efficiency assessment, with RNA elution for payload quantification.
• Circular dichroism (CD), differential scanning calorimetry (DSC), and gel electrophoresis for stability analysis.
• In vitro mRNA expression and translation assays using various reporter genes and COVID spike protein.

Main Results:

• High RNA loading efficiency (up to 95-98%) achieved with protamine coating.
• Significant increase in RNA melting point and enhanced thermal stability demonstrated.
• RNA integrity and comigration observed after storage at various temperatures.
• ZnO-protamine-mRNA samples retained high expression activity and functional translation in vitro and in cells.

Conclusions:

• Protamine-coated zinc oxide nanoparticles effectively stabilize RNA, enhancing its thermal stability and payload delivery.
• These nanoparticles demonstrate high loading efficiency and maintain functional activity after thermal stress.
• The findings support the preclinical development of ZnO-protamine-mRNA as a promising platform for RNA-based vaccines and therapeutics.