Synthesis of cell penetrating peptide sterol coupler and its liposome study on S-mRNA.

In order to overcome the current LNP-mRNA delivery system's weakness of poor stability and rapid degradation by nuclease, a novel chol-CGYKK molecule and then the new phospholipid liposome were designed and prepared. A solid phase approach synthesized CGYKK and connected it to cholesterol via a disulfide linker to form the desired chol-CGYKK. Four formulated samples with different proportions of excipients were prepared by freeze-drying cationic liposomes and packaged S-mRNA. The stability test shows that after six months at 4 °C, the encapsulation rate of this novel phospholipid liposome was still approximately 90%, which would significantly improve the storage and transportation requirement. Transmission electron microscopy, atomic force microscopy, and scanning electron microscopy indicated that the liposomes were spherical and uniformly dispersed. On comparing the levels of mRNA protein expression of the four formulated samples, the S protein vaccine expression of formulated sample 1 was the highest. Uptake by vector cells for formulated sample 1 showed that compared to Lipo2000, and the transfection efficiency was 66.7%. Furthermore, the safety evaluation of the CGYKK and mRNA vaccine liposomes revealed no toxic effects. The in vivo study demonstrated that this novel mRNA vaccine had an immune response. However, it was still not as good as the LNP group right now, but its excellent physicochemical properties, stability, in vitro biological activity, and in vivo efficacy against SARS-CoV-2 provided new strategies for developing the next generation of mRNA delivery system.