Resolving Heterogeneity of Targeted Lipid Nanoparticles Through Solution-Based Biophysical Analyses

Area of Science:

• Biotechnology
• Nanomedicine
• Pharmacology

Background:

• Targeted lipid nanoparticles (tLNPs) facilitate cell-specific nucleic acid delivery via targeting ligands for receptor-mediated uptake.
• tLNPs offer potential for pregnancy-associated applications, requiring precise delivery to minimize maternal toxicity and ensure fetal safety.
• Current rational tLNP design is hindered by a lack of understanding regarding how physicochemical properties impact biological performance.

Purpose of the Study:

• To resolve nanoscale heterogeneity in targeted lipid nanoparticles (tLNPs) and understand its impact on biological performance.
• To investigate how ligand conjugation affects tLNP subpopulations, size, shape, composition, and abundance.
• To correlate specific tLNP subpopulations with targeted placental RNA delivery efficacy in vivo.

Main Methods:

• Utilized asymmetric flow field-flow fractionation integrated with in-line UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS).
• Analyzed protein conjugation effects on the internal nanostructure and heterogeneity of lipid nanoparticles (LNPs).
• Employed chemometric SAXS analyses to correlate resolved tLNP subpopulations with in vivo transfection outcomes.

Main Results:

• Protein conjugation increases tLNP heterogeneity, particularly with larger or multivalent ligands, while preserving internal lipid-RNA nanostructure.
• tLNPs with higher-avidity ligands demonstrated more effective targeted placental RNA delivery in mice, despite increased heterogeneity.
• SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlated with targeted placental transfection; bulk metrics associated with hepatic delivery.

Conclusions:

• A separation-coupled biophysical platform can resolve previously inaccessible tLNP subpopulations.
• Understanding tLNP subpopulations is crucial for rational engineering of targeted RNA therapeutics.
• Ligand design and resulting subpopulation characteristics significantly influence targeted delivery efficacy and biodistribution.