Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery

Abstract

Lipid nanoparticles (LNPs) are formulated using unmodified cholesterol. However, cholesterol is naturally esterified and oxidized in vivo, and these cholesterol variants are differentially trafficked in vivo via lipoproteins including LDL and VLDL. We hypothesized that incorporating the same cholesterol variants into LNPs - which can be structurally similar to LDL and VLDL – would alter nanoparticle targeting in vivo. To test this hypothesis, we quantified how >100 LNPs made with 6 cholesterol variants delivered DNA barcodes to 18 cell types in wildtype, LDL R−/−, and VLDLR−/− mice that were both age-matched and female. By analyzing ~2,000 in vivo drug delivery data points, we found that LNPs formulated with esterified cholesterol delivered nucleic acids more efficiently than LNPs formulated with regular or oxidized cholesterol when compared across all tested cell types in the mouse. We also identified an LNP containing cholesteryl oleate that efficiently delivered siRNA and sgRNA to liver endothelial cells in vivo. Delivery was as - or more - efficient than the same LNP made with unmodified cholesterol. Moreover, delivery to liver endothelial cells was 3X more efficient than delivery to hepatocytes, distinguishing this oleate LNP from hepatocyte-targeting LNPs. RNA delivery can be improved by rationally selecting cholesterol variants, allowing optimization of nanoparticle targeting.

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

“…A case in point is that either through blocking or absence of NPC1, higher efficiency of nucleic acid delivery is achieved by preventing these particles from reaching non-productive sites. More recently, synthetic analogs of cholesterol have been used to show that its structure is important for effective in vivo delivery 59 . The importance of cholesterol has not gone unnoticed, stimulating decades of research on lipid-based delivery systems 18,60,61 .…”

Nanoparticles Containing Oxidized Cholesterol Deliver mRNA to the Liver Microenvironment at Clinically Relevant Doses

“…Fourth, LNPs and lipoproteins have similar size and composition. Finally, using JORDAN, we found that modified cholesterols alter LNP biodistribution [14] , however this study measured LNP biodistribution; it did not measure functional mRNA delivery. Given that less than 4% of exogenously delivered RNA escapes endosomes [15] and endosomal escape changes with cell type [16] , it is difficult to predict functional mRNA delivery using biodistribution.…”

Non-Viral Targeted Nucleic Acid Delivery: Apply Sequences for Optimization

“…They measured the functional Cre mRNA delivery of more than 250 LNPs to multiple cell types in vivo, and identified two LNPs that efficiently deliver siRNA, sgRNA and Cas9 mRNA to endothelial cells and mediate endothelial cell gene editing [ 195 ]. Combined with bioinformatics, the team performed in vivo directed evolution of RNA delivery, and identified one LNPs that effectively delivers siRNA and sgRNA to bone marrow endothelial cells (BMECs) in vivo [ 194 ]. Meanwhile, in vivo screening can help to reveal the relations between nanoparticle structures and activities.…”

“…After 3 days, ICAM-2 Low endothelial cells were isolated and the DNA barcodes within that population were sequenced. (Reproduced with permission from [ 194 ], American Chemical Society, 2018).…”