Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy.

Area of Science:

• Nanomedicine
• Biotechnology
• Materials Science

Background:

• Lipid nanoparticles (LNPs) are crucial carriers for mRNA therapeutics, with applications expanding beyond vaccines.
• Understanding the link between LNP formulation, internal structure, and transfection efficiency is vital for optimizing mRNA therapies.

Purpose of the Study:

• To investigate how different ionizable lipids, helper lipids, and polymer-grafted lipids (polysarcosine [pSar] or polyethylene glycol [PEG]) affect LNP internal structure and in vitro transfection efficiency.
• To evaluate the potential of pSar as a novel stealth moiety for enhancing mRNA LNP formulations.

Main Methods:

• Formulation of LNPs using varying ionizable lipids (DODMA, MC3), helper lipids (DOPE, DOPC), and polymer-grafted lipids (pSar, PEG).
• Physicochemical characterization including size, zeta potential, and RNA accessibility.
• Small-angle X-ray scattering (SAXS) to analyze internal LNP structure.
• In vitro transfection and cell-binding assays using human peripheral blood mononuclear cells (hPBMCs) with reporter mRNA.

Main Results:

• SAXS revealed distinct internal LNP structures based on ionizable and helper lipid composition.
• pSar moieties altered LNP surface morphology, increasing roughness.
• pSar-containing LNPs demonstrated generally higher transfection efficiency, with MC3/DOPE/pSar achieving the highest activity.

Conclusions:

• pSar is a promising stealth moiety for developing improved mRNA LNP products.
• SAXS is a valuable tool for rational LNP formulation development by elucidating structure-activity relationships.
• Optimizing LNP composition, particularly with pSar, can significantly enhance mRNA delivery efficiency.