Multiphasic lipid nanoparticles: structural heterogeneity drives endosomal bypass for enhanced RNA delivery

Area of Science:

• Biotechnology
• Nanomedicine
• Drug Delivery Systems

Background:

• Lipid nanoparticles (LNPs) are key for nucleic acid (NA) delivery, but endosomal escape is a major limitation, hindering cytosolic access.
• Current LNPs often rely on pH-triggered mechanisms, with limited efficiency in delivering NA to the cell's interior.

Purpose of the Study:

• To engineer structurally active LNPs with heterogeneous, multiphasic cores to improve NA delivery and endosomal escape.
• To investigate the role of internal nanostructure and phase separation in LNP-mediated intracellular delivery.

Main Methods:

• Utilized glycerol monooleate (GMO) and functional lipids to create multiphasic LNPs with topological mismatch.
• Employed cryo-electron microscopy and synchrotron small-angle X-ray scattering to analyze LNP internal structure.
• Assessed NA cytosolic release and transfection efficiency of the novel LNPs.

Main Results:

• Demonstrated composition-dependent multiphasic organization within LNPs, providing mechanistic insights.
• Showed that increased internal heterogeneity correlates with enhanced NA cytosolic release and transfection.
• Identified direct plasma membrane fusion as the delivery mechanism for multiphasic LNPs, bypassing endosomal entrapment.

Conclusions:

• Introduced a new LNP design strategy leveraging phase separation for enhanced NA delivery.
• Multiphasic LNPs offer a promising alternative to conventional LNPs by enabling efficient cytosolic release.
• This work provides a blueprint for engineering advanced NA delivery vectors with improved intracellular performance.