3 Citations
Nucleic acid nanoparticles NANPs fabricated by using DNA origami are an emerging delivery vector for nucleic acid therapeutics Despite their advantages over other nanomaterials that include controlled spatial presentation of targeting ligands such as lipids and sugars understanding their cell targeting and uptake mechanisms remains limited Here we investigated NANP cellular targeting uptake and delivery of small interfering RNAs siRNAs to liver and neuronal cell models in vitro Using a rational design approach we targeted NANPs to two clinically validated receptors the asialoglycoprotein receptor ASGPR and the low-density lipoprotein receptor LDLR respectively using GalNAc and lipidation We systematically evaluated how ... More
Nucleic acid nanoparticles (NANPs) fabricated by using DNA origami are an emerging delivery vector for nucleic acid therapeutics. Despite their advantages over other nanomaterials that include controlled spatial presentation of targeting ligands such as lipids and sugars, understanding their cell targeting and uptake mechanisms remains limited. Here, we investigated NANP cellular targeting, uptake, and delivery of small interfering RNAs (siRNAs) to liver and neuronal cell models in vitro. Using a rational design approach, we targeted NANPs to two clinically validated receptors, the asialoglycoprotein receptor (ASGPR) and the low-density lipoprotein receptor (LDLR), respectively, using GalNAc and lipidation. We systematically evaluated how the ligand valency, interligand spacing, linker length, and ligand chemistry affected NANP association with on- and off-target liver cell types, revealing the relative roles of the biomolecular corona, receptor engagement, and endocytosis in these targeting strategies. We found that lipidation enhanced NANP uptake into HepG2 cells, a model cell line for hepatocytes, by promoting apolipoprotein recruitment, LDLR engagement, and clathrin-mediated endocytosis and also increased association with nonparenchymal cells. HepG2 uptake was further improved by conjugating NANPs to lipids with higher valency provided that lipids were adequately displayed away from the surface of NANP edges with more lipophilic lipids yielding greater cell association. We then benchmarked the potential for NANPs to deliver siRNAs to HepG2 cells in comparison with lipid nanoparticle and conjugate technologies and explored lipid functionalization as a strategy for nonhepatic NANP targeting to model neuronal cells. Overall, this study advances the foundational understanding of how clinically relevant targeting ligands mediate NANP interactions with both on- and off-target liver cell types in vitro, offering insights into potential design criteria for nucleic acid therapeutic delivery. Less
Nucleic acid nanoparticles NANPs fabricated by using the DNA origami method have broad utility in materials science and bioengineering Their site-specific heterovalent functionalization with secondary molecules such as proteins or fluorophores is a unique feature of this technology that drives its utility Currently however there are few chemistries that enable fast efficient covalent functionalization of NANPs with a broad conjugate scope and heterovalency To address this need we introduce synthetic methods to access inverse electron-demand Diels Alder chemistry on NANPs We demonstrate a broad conjugate scope characterize application-relevant kinetics and integrate this new chemistry with strain-promoted azide alkyne cycloaddition chemistry ... More
Nucleic acid nanoparticles (NANPs) fabricated by using the DNA origami method have broad utility in materials science and bioengineering. Their site-specific, heterovalent functionalization with secondary molecules such as proteins or fluorophores is a unique feature of this technology that drives its utility. Currently, however, there are few chemistries that enable fast, efficient covalent functionalization of NANPs with a broad conjugate scope and heterovalency. To address this need, we introduce synthetic methods to access inverse electron-demand Diels–Alder chemistry on NANPs. We demonstrate a broad conjugate scope, characterize application-relevant kinetics, and integrate this new chemistry with strain-promoted azide–alkyne cycloaddition chemistry to enable heterovalent click reactions on NANPs. We applied these chemistries to formulate a prototypical chemical countermeasure against chemical nerve agents. We envision this additional chemistry finding broad utility in the synthetic toolkit accessible to the nucleic acid nanotechnology community. Less
Nucleic acid nanoparticles NANPs are increasingly used in preclinical investigations as delivery vectors Tools that can characterize assembly and assess quality will accelerate their development and clinical translation Standard techniques used to characterize NANPs like gel electrophoresis lack the resolution for precise characterization Here we introduce the use of charge detection mass spectrometry CD-MS to characterize these materials Using this technique we determined the mass of NANPs varying in size shape and molecular mass NANPs varying in production quality due to formulations lacking component oligonucleotides and NANPs functionalized with protein and nucleic acid-based secondary molecules Based on these demonstrations CD-MS ... More
Nucleic acid nanoparticles (NANPs) are increasingly used in preclinical investigations as delivery vectors. Tools that can characterize assembly and assess quality will accelerate their development and clinical translation. Standard techniques used to characterize NANPs, like gel electrophoresis, lack the resolution for precise characterization. Here, we introduce the use of charge detection mass spectrometry (CD-MS) to characterize these materials. Using this technique, we determined the mass of NANPs varying in size, shape, and molecular mass, NANPs varying in production quality due to formulations lacking component oligonucleotides, and NANPs functionalized with protein and nucleic acid-based secondary molecules. Based on these demonstrations, CD-MS is a promising tool to precisely characterize NANPs, enabling more precise assessments of the manufacturing and processing of these materials. Less