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Yet, rational tLNP design is limited by an incomplete understanding of how physicochemical properties influence biological performance. Traditional analytical methods report only ensemble\u2010averaged properties, leaving the nanoscale heterogeneity of tLNPs unresolved. Here, we utilize asymmetric flow field\u2010flow fractionation integrated with in\u2010line UV spectral analysis, light scattering, and synchrotron small\u2010angle X\u2010ray scattering (AF4\u2010UV\u2010DLS\u2010MALS\u2010SAXS) to resolve ligand\u2010dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. Protein conjugation preserves the internal lipid\u2013RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent ligands. Despite increased heterogeneity, tLNPs functionalized with higher\u2010avidity ligands achieve more effective targeted placental RNA delivery in mice. Chemometric SAXS analyses reveal that only SAXS\u2010resolved tLNP subpopulations, not ensemble\u2010averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation\u2010coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and provides insights to inform rational engineering of next\u2010generation targeted RNA therapeutics.<\/jats:p>","DOI":"10.1002\/adma.73984","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T08:40:56Z","timestamp":1784623256000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Resolving Heterogeneity of Targeted Lipid Nanoparticles Through Solution\u2010Based Biophysical Analyses"],"prefix":"10.1002","volume":"38","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6455-8183","authenticated-orcid":false,"given":"Hannah C.","family":"Geisler","sequence":"first","affiliation":[{"name":"Department of Bioengineering University of Pennsylvania  Philadelphia Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah 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