Novel Bivalent Ionizable Cationic Lipids For Efficient Delivery Of MRNA

As the third-generation vaccine technology,mRNA vaccine has received more and more attention in the COVID-19 pandemic,and has become the COVID-19 vaccine with the highest penetration rate and protection rat.mRNA is easily degraded in the external environment,thereby affecting translation and expression efficiency.At the same time,mRNA needs to pass through the "extracellular barrier" outside the cell and the "endosome escape" inside the cell before reaching the cell to function.Therefore,it is necessary to use delivery vectors to deliver mRNA to target cells.Lipid nanoparticles(LNPs)are currently the most advanced and mainstream mRNA delivery vector.The two mRNA COVID-19 vaccines developed by Moderna and Bio NTech both use LNPs delivery technology.LNPs consists of four components: ionizable cationic lipids,cholesterol,helper lipids and PEG-lipids,among which ionizable cationic lipids have high patent barriers.Breaking through patent restrictions to achieve independent research and development is a hot spot and difficult point in the field of mRNA delivery research in China.In this paper,based on the properties of ionizable cationic lipids,16 new bivalent ionizable cationic lipids in 4 series were designed and synthesized using benzene ring as the lipid molecular skeleton.Among them,in the A and B series lipid molecules,the cation is connected to the benzene ring skeleton by carbamate bond,and finally the ionizable headgroup and the hydrophobic tail group are connected by click chemistry;in the C and D series lipid molecules,the cation is connected to the benzene ring skeleton by ester bond,and the ionizable headgroup is connected to the hydrophobic tail group by ether bond.All target lipid molecules were structurally identified by H-NMR and highresolution mass spectrometry.The synthesized new cationic lipids were prepared into corresponding lipid nanoparticles by solvent injection method.The particle size and Zeta potential were characterized by nanoparticle size and potential analyzer,and the morphological structure was characterized by transmission electron microscope.The results showed that the nanoparticles prepared by A3 and A4 had good physical and chemical properties,the particle sizes were 198.5 nm and 192.1 nm,the dispersion coefficients were0.146 and 0.113,and the Zeta potentials were 1.90 m V and-4.43 m V.Lipid nanoparticles were screened in vitro by encapsulating EGFP-mRNA.The results showed that A3 and A4 had strong transfection ability on He La and 293 T cells.At the same time,the nanoparticles composed of A3 and A4 with different N/P were further evaluated.The results showed that A3 and A4 had the best transfection ability when N/P=4,the transfection ability of them was further verified in vitro transfection by encapsulating Luciferase-mRNA,then the cytotoxicity detection experiment shows that the lipid molecule A3 and A4 have no obvious cytotoxicity.In order to verify the delivery effect of lipid molecules at the animal level,A3 was selected for transfection experiments in vivo by encapsulating Luciferase-mRNA and SARS-Co V2-Spike-mRNA.The results showed that A3 lipid nanoparticles can effectively deliver mRNA in animals and express related proteins at high levels.