Rational preparation and application of a mRNA delivery system with cytidinyl/cationic lipid.

The messenger RNA (mRNA)-based therapy, especially mRNA vaccines, has shown its superiorities in versatile design, rapid development and scale production, since the outbreak of coronavirus disease 2019 (COVID-19). Although the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines had been approved for application, unexpected adverse events were reported to be most likely associated with the mRNA delivery systems. Thus, the development of mRNA delivery system with good efficacy and safety remains a challenge. Here, for the first time, we report that the neutral cytidinyl lipid, 2-(4-amino-2-oxopyrimidin-1-yl)-N-(2,3-dioleoyl-oxypropyl) acetamide (DNCA), and the cationic lipid, dioleoyl-3,3'-disulfanediylbis-[2-(2,6-diaminohexanamido)] propanoate (CLD), could encapsulate and deliver the COVID-19 mRNA-1096 into the cytoplasm to induce robust adaptive immune response. In the formulation, the molar ratio of DNCA/CLD to a single nucleotide of COVID-19 mRNA-1096 was about 0.9: 0.5: 1 (the N/P ratio was about 7: 1). The DNCA/CLD-mRNA-1096 lipoplexes were rationally prepared by the combination of the lipids DNCA/CLD with the aqueous mRNA solution under mild sonication to stimulate multiple interactions, including H-bonding, π-stacking and electrostatic force between the lipids and the mRNA. After intramuscular applications of the DNCA/CLD-mRNA-1096 lipoplexes, robust neutralizing antibodies and long-lived Th1-biased SARS-CoV-2-specific cell immunity were detected in the immunized mice, thus suggesting the DNCA/CLD a promising mRNA delivery system. Moreover, our study might also inspire better ideas for developing mRNA delivery systems.