Structural and functional stability of lyophilized nonencapsulated mRNA synthesized in vitro during long-term storage

INTRODUCTION. The low physicochemical stability of mRNA and the dependence of current mRNA vaccines on the successful encapsulation into lipid nanoparticles and adherence to mandatory cold chain requirements limit their storage and transportation. A promising approach is the lyophilization of nonencapsulated mRNA with its subsequent delivery by jet injection. However, the preservation of its structure and functional activity during long-term storage has been insufficiently studied.

AIM. To evaluate the structural and functional stability of lyophilized nonencapsulated mRNA during long-term storage and its immunogenicity as a component of a trivalent mRNA vaccine against seasonal influenza administered to mice by jet injection.

MATERIALS AND METHODS. mRNA encoding green fluorescent protein (GFP) was lyophilized with various cryoprotectants and stored at −20, 4, and 20 °C. Its functional activity was determined by GFP expression after transfection of HEK293 cells, and its integrity was analyzed by microchip electrophoresis. The immunogenicity of the lyophilized and nonlyophilized mRNA-Vector-Flu vaccine was evaluated in female inbred BALB/c mice after two jet immunizations by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HAI) assay, virus neutralization assay, and interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay.

RESULTS. Based on the results of a preliminary screening, a 10% trehalose formulation was selected for further studies because it preserved the functional activity of GFP mRNA at the level closest to that of the control preparation. The lyophilized GFP mRNA retained its functional activity after 12 months of storage at all tested temperatures; no statistically significant differences were found compared with the mRNA synthesized immediately before use (p>0.05). The major mRNA fraction was preserved, although minor fractions of partially degraded mRNA were detected after 6–12 months, particularly at 20 °C. After 3 months of storage of the vaccine at 4 °C, the geometric mean antibody titers against hemagglutinin subtype 1 (H1), hemagglutinin subtype 3 (H3), and influenza B hemagglutinin (HB) were 1:382,000, 1:628,000, and 1:273,000, respectively, compared with 1:710,000, 1:628,000, and 1:328,000 in the nonlyophilized vaccine group. HAI titers were 1:37–1:80, virus-neutralizing antibody titers were 1:50–1:90; and the numbers of IFN-γ-secreting cells were 440 and 500 spot-forming cells (SFC) per 1×10⁶ splenocytes. No significant between-group differences were identified.

CONCLUSIONS. Lyophilization with 10% trehalose preserved the functional activity of nonencapsulated mRNA for 12 months despite signs of partial degradation at 20 °C. After 3 months of storage at 4 °C, the trivalent vaccine retained its ability to induce humoral and T-cell immune responses in mice. These findings support the potential of combining lyophilization and jet injection for the development of stable mRNA vaccines.

1. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines — A new era in vaccinology. Nat Rev Drug Discov. 2018;17(4):261–79. https://doi.org/10.1038/nrd.2017.243

2. Chaudhary N, Weissman D, Whitehead KA. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat Rev Drug Discov. 2021;20(11):817–38. https://doi.org/10.1038/s41573-021-00283-5

3. Chheda U, Pradeepan S, Esposito E, et al. Factors affecting stability of RNA — Temperature, length, concentration, pH, and buffering species. J Pharm Sci. 2024;113(2):377–85. https://doi.org/10.1016/j.xphs.2023.11.023

4. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078–94. https://doi.org/10.1038/s41578-021-00358-0

5. Crommelin DJA, Anchordoquy TJ, Volkin DB, et al. Addressing the cold reality of mRNA vaccine stability. J Pharm Sci. 2021;110(3):997–1001. https://doi.org/10.1016/j.xphs.2020.12.006

6. Oude Blenke E, Örnskov E, Schöneich C, et al. The storage and inuse stability of mRNA vaccines and therapeutics: Not a cold case. J Pharm Sci. 2023;112(2):386–403. https://doi.org/10.1016/j.xphs.2022.11.001

7. Packer M, Gyawali D, Yerabolu R, et al. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat Commun. 2021;12(1):6777. https://doi.org/10.1038/s41467-021-26926-0

8. Wang C, Tang X, Jiang C, et al. Intradermal delivery of SARS-CoV-2 RBD3-Fc mRNA vaccines via a needle-free injection system induces robust immune responses in rats. Front Immunol. 2025;16:1530736. https://doi.org/10.3389/fimmu.2025.1530736

9. Abbasi S, Matsui-Masai M, Yasui F, et al. Carrier-free mRNA vaccine induces robust immunity against SARS-CoV-2 in mice and non-human primates without systemic reactogenicity. Mol Ther. 2024;32(5):1266–83. https://doi.org/10.1016/j.ymthe.2024.03.022

10. Kisakov DN, Karpenko LI, Kisakova LA, et al. Jet injection of naked mRNA encoding the RBD of the SARS-CoV-2 spike protein induces a high level of a specific immune response in mice. Vaccines (Basel). 2025;13(1):65. https://doi.org/10.3390/vaccines13010065

11. Sharabrin SV, Ilyichev AA, Kisakov DN, et al. Needle-free jet-delivered mRNA-vaccine encoding influenza A(H1N1)pdm09 hemagglutinin protects mice from lethal virus infection. Mol Biol. 2025;59(3):376–89. https://doi.org/10.1134/S0026893325700062

12. Yakovlev VA, Litvinova VR, Rudometova NB, et al. Immunogenic and protective properties of mRNA vaccine encoding hemagglutinin of avian influenza A/H5N8 virus, delivered by lipid nanoparticles and needle-free jet injection. Vaccines (Basel). 2025;13(8):883. https://doi.org/10.3390/vaccines13080883

13. Muramatsu H, Lam K, Bajusz C, et al. Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine. Mol Ther. 2022;30(5):1941–51. https://doi.org/10.1016/j.ymthe.2022.02.001

14. Suzuki Y, Miyazaki T, Muto H, et al. Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates. Mol Ther Nucleic Acids. 2022;30:226–40. https://doi.org/10.1016/j.omtn.2022.09.017

15. Khan MFH, Baudin F, Sudalaiyadum Perumal A, Kamen AA. Freeze-drying of mRNA-LNPs vaccines: A review. Vaccines (Basel). 2025;13(8):853. https://doi.org/10.3390/vaccines13080853

16. Arte KS, Chen M, Patil CD, et al. Recent advances in drying and development of solid formulations for stable mRNA and siRNA lipid nanoparticles. J Pharm Sci. 2025;114(2):805–15. https://doi.org/10.1016/j.xphs.2024.12.013

17. Sharabrin SV, Krasnikova SI, Kisakov DN, et al. A naked lyophilized mRNA vaccine against seasonal influenza, administered by jet injection, provides a robust response in immunized mice. Vaccines (Basel). 2026;14(1):56. https://doi.org/10.3390/vaccines14010056

18. Gross FL, Bai Y, Jefferson S, et al. Measuring influenza neutralizing antibody responses to A(H3N2) viruses in human sera by microneutralization assays using MDCK-SIAT1 cells. J Vis Exp. 2017;(129):56448. https://doi.org/10.3791/56448

19. Gonzalez TJ, Boothby TC. Properties governing dry-state stability of RNA in amorphous sugar formulations. Commun Mater. 2025;6:129. https://doi.org/10.1038/s43246-025-00850-y

20. Liu XH, Song HP, Tao LL, et al. Trehalose-loaded LNPs enhance mRNA stability and bridge the in vitro–in vivo efficacy gap. npj Vaccines. 2025;10:201. https://doi.org/10.1038/s41541-025-01253-3

21. Khan MFH, Sudalaiyadum Perumal A, Kamen AA. Investigation on a freeze-drying process for long-term stability of mRNA-LNPs. Vaccines (Basel). 2026;14(3):242. https://doi.org/10.3390/vaccines14030242