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

Area of Science:

• Biotechnology
• Nanomedicine
• Drug Delivery

Background:

• Targeted lipid nanoparticles (tLNPs) facilitate cell-specific nucleic acid delivery via receptor-mediated uptake.
• tLNPs hold potential for pregnancy applications, demanding precise delivery to ensure maternal safety and fetal well-being.
• Current understanding of physicochemical properties' impact on tLNP biological performance is limited, hindering rational design.

Purpose of the Study:

• To investigate how ligand conjugation influences the heterogeneity and biological performance of targeted lipid nanoparticles (tLNPs).
• To develop and apply a comprehensive analytical platform for resolving tLNP subpopulations.
• To correlate specific tLNP characteristics with targeted delivery efficiency in vivo.

Main Methods:

• Utilized asymmetric flow field-flow fractionation coupled with UV-Vis spectroscopy, dynamic light scattering, multi-angle light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS).
• Analyzed ligand-dependent tLNP subpopulations based on size, shape, composition, and abundance.
• Employed chemometric SAXS analysis to link resolved subpopulations with in vivo delivery outcomes.

Main Results:

• Protein conjugation increased tLNP heterogeneity, particularly with larger or multivalent ligands, while preserving internal nanostructure.
• tLNPs with higher-avidity ligands demonstrated enhanced targeted placental RNA delivery in a murine model.
• SAXS-resolved subpopulations, not ensemble-averaged properties, correlated with placental transfection, while bulk metrics predicted hepatic delivery.

Conclusions:

• The developed separation-coupled biophysical platform resolves previously inaccessible tLNP subpopulations.
• Ligand characteristics significantly influence tLNP heterogeneity and in vivo targeting efficiency.
• Insights gained can inform the rational design of advanced targeted RNA therapeutics for improved safety and efficacy.