Biology subjects

Safford, H. C.

Publications and source records attributed to Safford, H. C..

5 recordsLinked to original sources

bioRxiv2026

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

Targeted lipid nanoparticles (tLNPs) represent the next frontier in nucleic acid therapeutics, enabling cell-specific delivery through covalent attachment of targeting ligands that drive receptor-mediated uptake. tLNPs are particularly promising for pregnancy-associated applications where precise on-target delivery is required to minimize maternal toxicity and protect fetal health. Yet, their rational design is limited by an incomplete understanding of how tLNP physicochemical properties influence biological performance. Conventional LNPs already exhibit pronounced heterogeneity in size, composition, and RNA loading, which is further amplified in tLNPs by variability in ligand attachment and surface density. Because traditional analytical methods report only ensemble-averaged properties, the nanoscale diversity of tLNPs remains unresolved. Here, we find that tLNP functional behavior is governed by previously inaccessible, structurally distinct tLNP subpopulations that are not captured by bulk measurements. We utilize 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) to resolve ligand-dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. We find that protein conjugation preserves the internal lipid-RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent targeting ligands. Despite increased heterogeneity, tLNPs functionalized with higher-avidity ligands achieve more effective targeted placental RNA delivery in mice, suggesting that binding avidity can offset the functional consequences of polydispersity. Chemometric SAXS analyses reveal that only SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk-derived physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation-coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and demonstrates that subpopulation nanoscale structure, rather than bulk-averaged properties, dictates targeted RNA delivery. These insights provide a mechanistic foundation for rational engineering of next-generation precision targeted RNA LNP therapeutics.

bioengineering↗

bioRxiv2025

Bioinspired Oxidized mRNA Lipid Nanoparticles for Ex Vivo Engineering of Chimeric Antigen Receptor Macrophages Targeting Solid Tumors

Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The composition of the top LNP was subsequently optimized using an orthogonal design of experiments (DoE) and physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding a particle that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E (ApoE) independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform can potentially be utilized to engineer a range of human CAR-M immunotherapies to treat various types of solid tumors.

bioengineering↗

bioRxiv2025

High-throughput in vivo screening using barcoded mRNA identifies lipid nanoparticles with extrahepatic tropism for cancer immunotherapy

Interest continues to grow in the use of mRNA vaccines for cancer immunotherapy. While effective for immunization against infectious diseases, current clinical lipid nanoparticle (LNP) formulations used for mRNA delivery suffer from off-target accumulation, poor immune transfection, and reactogenicity, limiting their application to cancer immunotherapy. Development of new mRNA LNPs is severely bottlenecked by the LNP discovery process, which is historically low-throughput due to reliance on low-plexity measurements. Here, we develop a next-generation high-throughput in vivo mRNA LNP screening platform based on barcoded mRNA (b-mRNA). Using this b-mRNA screening platform to simultaneously evaluate 122 LNPs, we identify novel LNP formulations capable of potent hepatic and extrahepatic transfection. We employ novel biochemical characterization techniques to analyze nanoparticle protein corona formation with single-particle resolution and gain insight into the influence of protein adsorption on hepatic and splenic transfection. We evaluate a lead LNP candidate for therapeutic cancer vaccination in a syngeneic mouse model of melanoma and demonstrate a significant reduction in tumor burden and increase in survival compared to a clinical mRNA LNP formulation. Together, our results demonstrate the value of advanced LNP screening and characterization techniques for the development of next-generation mRNA therapeutics and vaccines.

bioengineering↗

bioRxiv2021

A predictive microfluidic model of human glioblastoma to assess trafficking of blood-brain barrier penetrant nanoparticles

The blood-brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood-brain barrier vasculature. Here, we report a vascularized human glioblastoma (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood-brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood-brain barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics. Significance StatementThe blood-brain barrier represents a major therapeutic challenge for the treatment of glioblastoma, and there is an unmet need for in vitro models that recapitulate human biology and are predictive of in vivo response. Here we present a new microfluidic model of vascularized glioblastoma featuring a tumor spheroid in direct contact with self-assembled vascular networks comprised of human endothelial cells, astrocytes, and pericytes. This model was designed to accelerate the development of targeted nanotherapeutics, and enabled rigorous assessment of a panel of surface-functionalized nanoparticles designed to exploit a receptor overexpressed in tumor-associated vasculature. Trafficking and efficacy data in the in vitro model compared favorably to parallel in vivo data, highlighting the utility of the vascularized glioblastoma model for therapeutic development.

bioengineering↗

bioRxiv2021

Massively parallel pooled screening reveals genomic determinants of nanoparticle-cell interactions

To accelerate the translation of cancer nanomedicine, we hypothesize that integrated genomic screens will improve understanding of the cellular processes governing nanoparticle trafficking. We developed a massively parallel high-throughput screening method leveraging barcoded, pooled cancer cell lines annotated with multi-omic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. This approach identified both the materials properties and cell-intrinsic features mediating nanoparticle-cell association. Coupling the data with machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers, including gene expression of SLC46A3. We engineered cell lines to validate SLC46A3 as a biomarker whose expression inversely predicts liposomal nanoparticle uptake both in vitro and in vivo. We further demonstrated the predictive capabilities extend beyond liposomal nanoparticles, regulating both uptake and transfection efficacy of solid lipid nanoparticles. Our work establishes the power of massively parallel pooled cell screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations for specific patient populations.

bioengineering↗