Exploring\nthe Effects of Incorporating Different Bioactive\nPhospholipids into Messenger Ribonucleic Acid Lipid Nanoparticle (mRNA\nLNP) Formulations

The current rapid advancement in ribonucleic acid (RNA)\ntherapeutics\nresearch depends on innovations in drug delivery, especially the development\nof a lipid-nanoparticle (LNP)-based system. The conventional LNP formulation\ntypically contains four components, including an ionizable cationic\nlipid, a phospholipid, cholesterol or a cholesterol derivative, and\npoly(ethylene glycol) (PEG)-lipid, with each contributing to the formulation’s\noverall stability and effectiveness. Among these four types of lipids,\nthe phospholipid component is often known to provide structural support\nfor the nanoparticles but is also a class of bioactive molecules with\nstrong cell signaling potential. This study explores the possibility\nof incorporating some known structurally related bioactive phospholipids\nas the fifth component of a conventional four-component LNP formulation\nand assesses the impacts of such an approach on the physicochemical\nproperties and biological functions of the mRNA LNP formulation. We\nscreened a library of mRNA LNP formulations containing 7 different\nstructurally related bioactive phospholipids at molar concentrations\nof 5%, 15% and 30% in addition to a conventional four-component LNP\nformulation (base). We observed differences in physicochemical properties\nbetween the mRNA LNP formulations that could be attributed to both\nthe types of phospholipids examined and the molar concentrations used.\nCryo-EM analysis revealed structural similarity between the Base formulation\nand the other formulations. We also characterized the protein expression\nlevel in HeLa cells and picked up a distinct cytokine panel signature\nfor each formulation in human peripheral blood mononuclear cells (hPBMCs).\nFurther immunophenotyping analysis showed that most cells that were\ntransfected were CD4+ T cells, and the addition of the different bioactive\nphospholipids slightly altered cellular tropism. This exploratory\nstudy illustrates how adding the bioactive phospholipid can be used\nto modulate the LNP function, further expanding the design space for\nRNA LNP formulations and potentiating LNPs for use as RNA therapeutics.