Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart.

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

The Transcriptional Response to Lung-Targeting Lipid Nanoparticles in Vivo.

Lipid nanoparticles (LNPs) have delivered RNA to hepatocytes in patients, underscoring the potential impact of nonliver delivery. Scientists can shift LNP tropism to the lung by adding cationic helper lipids; however, the biological response to these LNPs remains understudied. To evaluate the hypothesis that charged LNPs lead to differential cellular responses, we quantified how 137 LNPs delivered mRNA to 19 cell types in vivo. Consistent with previous studies, we observed helper lipid-dependent tropism. After identifying and individually characterizing three LNPs that targeted different tissues, we studied the in vivo transcriptomic response to these using single-cell RNA sequencing. Out of 835 potential pathways, 27 were upregulated in the lung, and of these 27, 19 were related to either RNA or protein metabolism. These data suggest that endogenous cellular RNA and protein machinery affects mRNA delivery to the lung in vivo.

Self-Assembled Gold Nanoparticle-Fluorescent Protein Conjugates as Platforms for Sensing Thiolate Compounds via Modulation of Energy Transfer Quenching.

The ability of Au and other metal nanostructures to strongly quench the fluorescence of proximal fluorophores (dyes and fluorescent proteins) has made AuNP conjugates attractive for use as platforms for sensor development based on energy transfer interactions. In this study, we first characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-histidine coordination, where parameters such as NP size and number of attached proteins are varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ∼95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further exploited these findings to develop a solution phase sensing platform targeting thiolate compounds. Energy transfer (ET) was employed as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. Our results show that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction, and type of thiol derivatives used. Further analysis of the PL recovery data provides a measure for the equilibrium dissociation constant (Kd-1) for these compounds. These findings combined indicate that the AuNP-fluorescent protein conjugates may offer a potentially useful platform for thiol sensing both in solution and in cell cultures.