Novel siloxane-incorporated LNPs for tissue-specific mRNA delivery

Advances in mRNA-based vaccines, protein-replacement therapy, and gene editing have, in part, been enabled by synthesis and screening of ionizable lipids for improved lipid nanoparticle (LNP) formulations. In the bloodstream, ionizable lipids are not charged, thus preventing toxicity; inside the target cell, they become positively charged, triggering the release of the mRNA payload.?

Tissue-specific targeting of mRNA-LNPs is desirable for systemic administration. Recently, novel silicon (Si)-containing ionizable lipids for organ-specific mRNA-LNP delivery were reported by Xue et al. in Nature Nanotechnology. Interestingly, Si has been virtually ignored as an atomic component in LNPs (Holland et al.). Compounds with lipid characteristics are, in general, called lipidoids.?

The publication by Xue et al., which reports use of TriLink CleanCap? Cre mRNA,? OVA mRNA, and Cas9 mRNA, each modified with 5-methoxyuridine (5moU), has already received more than 8,500 views since its publication on October 1, 2024. As outlined below, the engineered siloxane-incorporated LNPs, termed SiLNPs, control in vivo mRNA delivery to either liver, lung, or spleen in mice.??

Design and synthesis of siloxane ionizable lipidoids for SiLNP screening??

Combinatorial reactions between siloxane amines and either epoxide, amide, or ester reagents bearing hydrocarbon chains led to the synthesis of 252 siloxane-containing ionizable lipidoids. This is exemplified in Figure 1 for reaction of a diamino siloxane with either an amide or epoxide reagent, each bearing a C12 hydrocarbon chain; in esters, the HN-C=O is replaced by O-C=O. With either the amide or the epoxide, the ratios of reagents used lead to attachment of four C12-chains to the diamino siloxane, i.e., two C12-chains are added to each of the two NH2 positions.??????

The 252 siloxane-containing ionizable lipidoids represented a large structural diversity, due to the use of either diamines (Figure 1) or mono-, tri-, and tetra-amines. Additionally, the siloxanes contained either two silicon atoms (Figure 1) or more, while the hydrocarbon chains were varied from C6 to C18.??

To evaluate the structure-activity relationships of the 252 siloxane lipidoids for mRNA delivery, SiLNPs were formulated with firefly luciferase (FLuc) mRNA using fixed molar ratios of each siloxane lipidoid and the other LNP components, namely, the phospholipid DOPE, cholesterol, and a lipid-anchored PEG (C14PEG2K). An FDA-approved LNP formulation comprised of the ionizable lipid MC3 was used as a non-siloxane positive control. Fluc expression following transfection of human liver carcinoma (HepG2) cells was used to rank-order delivery efficiency in vitro.?

SiLNPs enable tissue-specific mRNA delivery in vivo?

The top 50 SiLNPs from in vitro screening in HepG2 cells were selected for comparative in vivo delivery of FLuc mRNA in mice. Of these lead candidates, 14 failed to show significant luciferase expression in vivo and were not studied further. The remaining 36 SiLNPs were individually administered intravenously (i.v.) to mice for quantitative analysis of Fluc bioluminescence 6 h post-injection. Of the five major organs examined — namely, heart, liver, spleen, lung, and kidney — selective delivery was found for liver, lung, and spleen.?

In brief, the top-performing liver-targeted SiLNP (liver-SiLNP) exhibited ~98% luciferase expression in liver with only ~2% distributed among the four other organs analyzed. For the best lung-specific SiLNP (lung-SiLNP) and spleen-specific SiLNP (spleen-SiLNP), the luciferase expression specificities were ~90% and ~70%, respectively. The molecular structures of these liver-, lung-, and spleen-SiLNPs are detailed in Xue et al. Further studies of each of these SiLNPs are discussed in what follows.?

Liver-SiLNPs for CRISPR-Cas9 editing in the liver?

To characterize the transfection of liver-cell types, the Cre-LoxP mouse (Ai14)model that expresses Lox-STOP-Lox tdTomato was used. In this widely used model, upon intracellular delivery of Cre mRNA, the translated Cre-recombinase protein deletes the STOP cassette and thereby activates expression of the fluorescent protein tdTomato.??

Following a single i.v. injection of TriLink CleanCap? Cre mRNA in liver-SiLNPs, flow cytometry demonstrated functional Cre mRNA delivery to ~35% of hepatocytes, ~70% of liver sinusoidal endothelial cells, and ~82% of Kupffer cells, all of which were higher than the delivery efficacy of the MC3 LNP control.?

To evaluate CRISPR-Cas9 genome editing in a therapeutic mouse model, liver-SiLNPs were formulated with TriLink CleanCap?? Cas9 mRNA and a single-guide (sg) RNA targeting the mouse liver TTR gene encoding transthyretin (TTR), which transports vitamin A and the hormone thyroxine throughout the body. TTR amyloidosis is a life-threatening disease characterized by progressive accumulation of misfolded TTR.?

Gene editing was quantified by examining serum TTR protein concentration and on-target editing through DNA sequencing, following a single i.v. injection at increasing doses, with corresponding MC3 LNPs as a positive control. Liver-SiLNPs mediated a dose-dependent knock-down of serum TTR that was ~2- to 3-fold greater than that of MC3 LNPs. Delivery by liver-SiLNPs led to editing as soon as 6 h post-injection, which increased at later time points and lasted for at least 56 days.??

Lung-SiLNPs for CRISPR-Cas9 editing in the lungs?

To explore the mechanism of lung targeting by lung-SiLNPs, Xue et al. used mass spectrometric proteomic methodology (Qui et al.) to identify and quantify the top 20 proteins bound to lung-SiLNPs following incubation in mouse plasma. Among the bound proteins, which are known as protein corona, vitronectin (Vtn) was the most abundant at ~16%, a 320-fold enrichment over its abundance in mouse plasma. Vtn can bind its cognate receptor, αvβ3 integrin, which is highly expressed by the pulmonary endothelium, thus providing a plausible explanation for lung targeting by lung-SiLNPs.??

To characterize transfected cell types in the lungs, TriLink CleanCap? Cre mRNA formulated in lung-SiLNPs was administered i.v. to Ai14 mice. After 3 days, flow cytometry indicated high specificity for lung endothelial cells (~88% tdTomato-positive). Immunostaining of the lungs showed that lung-LNPs mainly transfected the capillary endothelial cells, with low transfection of the large vessels and airway.?

Xue et al. next assessed in vivo delivery of TriLink CleanCap?? Cas9 mRNA and green fluorescent protein (GFP) sgRNA co-formulated in lung-SiLNPs for editing in the lungs of transgenic GFP mice upon daily i.v. administration for 4 days. Lung tissue removed 7 days after the final injection showed GFP knock-down in ~20% of endothelial cells and ~8% of epithelial cells, which was confirmed by immunostaining.?

Endothelial cells from the lungs were then sorted to evaluate the editing efficacy by real-time quantitative PCR (RT-qPCR), which demonstrated that GFP expression decreased ~2-fold after lung-SiLNP-mediated CRISPR-Cas9 editing compared to controls.??

To show the therapeutic editing potential of lung-SiLNPs, a classical Lewis lung carcinoma (LLC) tumor model in mice was used to demonstrate knock-down of vascular endothelial growth factor receptor 2 (VEGFR2) expression in lung endothelial cells for antiangiogenic cancer therapy. Mice bearing LLC tumors were systemically treated with lung-SiLNPs encapsulating Cas9 mRNA/VEGFR2 sgRNA, while Cas9 mRNA/scramble sgRNA-loaded lung-SiLNPs and buffer-treated groups were used as controls.??

One week after LLC-bearing mice had been treated 3-times with lung-SiLNPs encapsulating TriLink CleanCap?? Cas9 mRNA/VEGFR2 sgRNA, excised tissue exhibited an ~2-fold decrease in both VEGFR2 expression tumor burden compared to controls.?

Lung-SiLNPs enable endothelial repair for lung regeneration?

Owing to the potent pulmonary endothelium targeting of lung-SiLNPs, Xue et al. next evaluated the therapeutic potential of lung-SiLNPs for treating vascular-related diseases in the lungs. To do this, a viral infection lung vasculature damage model was established to investigate whether endothelial overexpression of fibroblast growth factor 2 (FGF2) would accelerate the recovery of lung function.??

Following i.v. administration of FGF2 mRNA in lung-SiLNPs, the expressed FGF2 protein, which was detected in bronchoalveolar lavage fluid (BALF), led to improved lung function, as evidenced by improved recovery of body weight and increased blood oxygen levels compared with the control groups. Histopathological evaluation of the lungs showed less inflammation and improved remodeling with FGF2 mRNA in lung-SiLNPs compared to controls.?

Spleen-SiLNPs for targeting splenic immune cells?

As reviewed elsewhere (Nie et al.), there is growing evidence that direct delivery of mRNA vaccines into the spleen confers substantially enhanced immune responses compared to local routes of administration. Transfection of splenic antigen-presenting cells, such as dendritic cells (DCs) and macrophages, would initiate robust long-lived antigen-specific CD8 T-cell responses, critical for anti-cancer vaccines. However, splenic delivery remains perhaps the biggest bottleneck to i.v. administration of mRNA vaccines.?

To characterize the transfection of spleen-cell types, Xue et al. used the above-mentioned Ai14 mouse model to measure tdTomato expression following i.v. administration of TriLink CleanCap? Cre mRNA delivery in spleen-SiLNPs. Flow cytometry of necropsy samples obtained 3 days post-injection led to tdTomato-positive quantification of T cells, B cells, DCs, and macrophages, with DCs and macrophages being the most prevalent.?

The possibility of siloxane-mediated adjuvant activity was ruled out by incubation of DCs with spleen-SiLNLPs containing TriLink CleanCap? OVA mRNA, which encodes ovalbumin, a widely used model antigen. Measured DC maturation was not statistically different than that of the controls.?

Concluding Comments?

While not discussed above, Xue et al. provide data demonstrating that the incorporation of a siloxane moiety into ionizable lipidoids enhances both the endocytosis and endosomal escape of mRNA-SiLNPs compared to non-siloxane LNP controls. These advantageous properties were correlated with the increased hydrophobicity and size of siloxane moieties.?

The remarkable liver-, lung-, and spleen-specificities found for the above mentioned SiLNPs underscores the importance of using non-traditional atomic elements, such as Si, to engineer structurally diverse libraries of ionizable lipidoids for screening.??