Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses.

Area of Science:

• Biotechnology
• Nanomedicine
• Drug Delivery

Background:

• Targeted lipid nanoparticles (tLNPs) enable cell-specific nucleic acid delivery via targeting ligands.
• Precise delivery is crucial for pregnancy applications to minimize maternal toxicity and protect fetal health.
• Current understanding of tLNP physicochemical properties' impact on biological performance is limited due to heterogeneity.

Purpose of the Study:

• To investigate how tLNP physicochemical properties and nanoscale heterogeneity influence biological performance.
• To resolve previously inaccessible, structurally distinct tLNP subpopulations.
• To establish a mechanistic foundation for rational engineering of targeted RNA LNP therapeutics.

Main Methods:

• Utilized asymmetric flow field-flow fractionation integrated with UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS).
• Separated tLNP subpopulations based on size, shape, composition, and relative abundance.
• Performed chemometric SAXS analyses to correlate subpopulation structure with in vivo delivery.

Main Results:

• Protein conjugation increased tLNP heterogeneity, especially with larger/multivalent ligands, while preserving internal nanostructure.
• Higher avidity ligands on tLNPs improved targeted placental RNA delivery in mice, offsetting polydispersity.
• SAXS-resolved tLNP subpopulations correlated with targeted placental transfection, unlike bulk-averaged properties associated with hepatic delivery.

Conclusions:

• tLNP functional behavior is governed by distinct subpopulations not captured by bulk measurements.
• Nanoscale structure of tLNP subpopulations, not ensemble-averaged properties, dictates targeted RNA delivery.
• This study provides a mechanistic basis for designing next-generation precision targeted RNA LNP therapeutics.