Quaternization drives spleen-to-lung tropism conversion for mRNA-loaded lipid-like nanoassemblies

Abstract

Background: As the overwhelming majority of advanced mRNA delivery systems are preferentially accumulated in the liver, there is an accelerating growth in the demand for the development of non-liver mRNA delivery platforms. Methods: In this study, we prepared cationic lipid-like nanoassemblies through a N-quaternizing strategy. Their physicochemical properties, in vitro mRNA delivery efficiency, and organ tropism in mice were investigated. Results: Introduction of quaternary ammonium groups onto lipid-like nanoassemblies not only enhances their mRNA delivery performance in vitro, but also completely alters their tropism from the spleen to the lung after intravenous administration in mice. Quaternized lipid-like nanoassemblies exhibit ultra-high specificity to the lung and are predominantly taken up by pulmonary immune cells, leading to over 95% of exogenous mRNA translation in the lungs. Such mRNA delivery carriers are stable even after more than one-year storage at ambient temperature. Conclusions: Quaternization provides an alternative method for design of new lung-targeted mRNA delivery systems without incorporation of targeting ligands, which should extend the therapeutic applicability of mRNA to lung diseases.

Keywords: lipid-like nanoassembly; lung targeting; quaternization; systemic mRNA delivery; ultra-high selectivity.

Figure 1

Schematic of tropism conversion of mRNA-loaded lipid-like nanoassemblies from the spleen to the lung via quaternization. The quaternized tB-UC18 derivative (qtB-UC18) in (B) was obtained through the N-alkylation reaction of secondary amines of tB-UC18 in (A) with iodomethane.

Figure 2

Optimization of qtB-UC18 LLNs for intracellular mRNA delivery. (A) Sequential optimization of formulation parameters in the order of N:P ratios, followed by molar ratios. (B) Sequential optimization of formulation parameters in the order of molar ratios, followed by N:P ratios. The formulation in (A) and (B) was expressed as qtB-UC18:DOPE:mRNA, where qtB-UC18:DOPE represented the molar ratio between qtB-UC18 and DOPE, and qtB-UC18:mRNA represented the N:P ratio between qtB-UC18 and mRNA. Luciferase activity of FLuc mRNA-loaded LLNs was normalized to the Lipo2k group. The light blue zone indicated N:P ratio optimization while the pink zone indicated molar ratio optimization. (C, D) The hydrodynamic size (C) and zeta potential (D) of qtB-UC18 LLNs formed through complexation between qtB-UC18 and mRNA. (E, F) The hydrodynamic size (E) and zeta potential (F) of qtB-UC18 LLNs formed through complexation between qtB-UC18/DOPE and mRNA. (G) Fluorescent images of cells incubated with eGFP mRNA-loaded qtB-UC18 LLNs for 48 h. Scale bar, 100 μm.

Figure 3

Cellular uptake and hemolytic activity of qtB-UC18 LLNs. (A) Representative flow cytometric histograms for analysis of cellular uptake mediated by different formulations. (B) The effects of endocytosis inhibitors and temperature on the cellular uptake of qtB-UC18 LLNs containing Cy5-labelled mRNA. The relative fluorescence intensity (RFI) of each group was normalized to the PBS (no inhibitor) group. (C) Hemolysis detection for single-component or dual-component carriers. The hemolytic activity was normalized to that of Triton X-100.

Figure 4

Delivery efficiency and specificity of formulations tested in the in vivo studies. (A) In vivo bioluminescence imaging of luciferase expression mediated by qtB-UC18 LLNs 4 h after intravenous injection. tB-UC18 LLNs served as a control. (B) Quantification of luciferase expression in each organ examined. (C) The percentage of luciferase expression in major organs.

Figure 5

The bio-distribution of fluorescently labeled LTNs and the protein corona adsorption onto LTNs. (A) The bio-distribution of DiD-incorporated LTNs in the heart (line 1), liver (line 2), spleen (line 3), lung (line 4), and kidney (line 5) 4 h after intravenous administration. (B) Flow cytometric analysis of the percentage of DiD positive cells for each cell populations in the lung tissues from mice receiving DiD-incorporated LTNs. (C) Identification of the top 20 most abundant proteins adsorbed to formulations by LC-MS/MS.

Figure 6

Stability of lung-targeted nanocarriers and nanoformulations. (A) The representative transmission electron micrograph showing the morphology of LTNs. (B) Assessment of the resistance of LTNs to RNase A-induced degradation through gel electrophoresis. LTN-encapsulated or free mRNA were incubated with RNase A at the indicated time points before electrophoresis. (C, D) Serum stability of LTNs determined by monitoring changes in turbidity (C) and hydrodynamic size (D). (E) The effect of temperature on the hydrodynamic size and polydispersity index of LTNs. (F) Assessment of the long-term stability of lung-targeted nanocarriers containing equimolar qtB-UC18 and DOPE after 200, 340, and 450 days of storage in an ethanol/water solvent at ambient temperature. The delivery performance was evaluated by encapsulating eGFP mRNA into stored nanocarriers and monitoring the green fluorescent signals originating from 293T cells after 24 h of treatment.