Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for RNA and have enabled the development of RNA-based drugs such as the mRNA COVID-19 vaccines. Functional delivery of mRNA by an LNP greatly depends on the inclusion of an ionizable lipid, and small changes to these lipid structures can significantly improve delivery. However, the structure–function relationships between ionizable lipids and mRNA delivery are poorly understood, especially for LNPs administered intramuscularly. Here, we show that the iterative design of a novel series of ionizable lipids generates key structure–activity relationships and enables the optimization of chemically distinct lipids with efficacy that is on-par with the current state of the art. We find that the combination of ionizable lipids comprising an ethanolamine core and LNPs with an apparent pKa between 6.6 and 6.9 maximizes intramuscular mRNA delivery. Furthermore, we report a nonlinear relationship between the lipid-to-mRNA mass ratio and protein expression, suggesting that a critical mass ratio exists for LNPs and may depend on ionizable lipid structure. Our findings add to the mechanistic understanding of ionizable lipids and demonstrate that hydrogen bonding, ionization behavior, and lipid-to-mRNA mass ratio are key design parameters affecting intramuscular mRNA delivery. We validate these insights by applying them to the rational design of new ionizable lipids. Overall, our iterative design strategy efficiently generates potent ionizable lipids. This hypothesis-driven method reveals structure–activity relationships that lay the foundation for the optimization of ionizable lipids in future LNP-RNA drugs. We foresee that this design strategy can be extended to other optimization parameters beyond intramuscular expression

Dendrimer-Inspired Nanomaterials for the <i>in Vivo</i> Delivery of siRNA to Lung Vasculature

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly­(amido amine) and poly­(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from <i>in vivo</i> screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer–lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics

Dendrimer-Inspired Nanomaterials for the <i>in Vivo</i> Delivery of siRNA to Lung Vasculature

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly­(amido amine) and poly­(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from <i>in vivo</i> screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer–lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics

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orbital symmetries and ionization dynamic

Delivery to human immune cells.

<p><b>A)</b> Human T cells and MDDCs were tested for delivery of cascade blue labeled 3kDa dextran, fluorescein labeled 70kDa dextran, and APC labeled IgG1. The representative histograms for a 30–4 (T cells) and 10–7 (MDDCs) device (left) and replicates across device designs (right) are displayed. <b>B)</b> SiRNA mediated knockdown of CD4 and DC-SIGN protein levels in CD4⁺ T cells and MDDCs respectively. <b>C)</b> Knockdown of CD4 expression in human regulatory T cells in response to treatment by a 30–4 device. Dead cells were excluded for delivery or knockdown analysis. <b>D)</b> Comparison of device performance in T cells to nucleofection by Amaxa. Protein expression 72hrs after siRNA delivery and cell viability after treatment are shown. <b>E)</b> Intracellular staining for the p24 antigen was used as an indicator of HIV infection level in treated human CD4⁺ T cells 24hrs after infection. In these studies, vif and/or gag, siRNA was delivered 24hrs prior to infection while CD4 siRNA was delivered 48hrs prior to infection. <b>F)</b> Median fluorescence intensity of the p24 antigen stain across repeats (min. N = 4) of the experimental conditions. Data are represented as mean + 1 standard error.</p

Delivery methodology and performance in mouse cells.

<p><b>A)</b> Illustration of device design and delivery mechanism. <b>B)</b> Illustration of the system setup and delivery procedure. <b>C)</b> Representative histograms of T cells, B cells and myeloid cells (CD11b⁺) treated by the CellSqueeze device to deliver APC-labeled IgG1. <b>D)</b> Delivery efficiency of Cascade blue-labeled 3 kDa dextran, fluorescein-labeled 70 kDa dextran, and APC-labeled IgG1. All results were measured by flow cytometry within an hour of treatment. Dead cells were excluded by propidium iodide staining. Viability is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118803#pone.0118803.s002" target="_blank">S2 Fig</a></b>. Data in <b>D)</b> (mean ± SD) are from 3 independent experiments. Untreated cells were not put through the device or exposed to the biomolecules. The ‘no device’ samples were incubated with the biomolecules, but were not treated by the device. This control is meant to account for surface binding, endocytosis and other background effects.</p