Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart

Abstract

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

Keywords: LNP; barcoding; mRNA; nanoparticle; scRNA-seq.

Fig. 1.

Four libraries containing LNPs with varied charge of cholesterol or helper lipid formed small and stable LNPs. (A and B) LNPs were formulated at eight different mole ratios of the four components by varying nine helper lipids and three cholesterols, creating 216 LNPs in total. (C) LNPs in each library (gray) formed small (50 to 200 nm) LNPs with the pool diameter (purple) being in the range of the pooled LNPs from each library, indicating no LNP aggregation. The (0 +) library consisted of LNPs with significantly larger diameter. The reported diameters include all eight tested molar ratios. One-way ANOVA, mean diameter of each library was compared to the mean diameter of every other library, ***P < 0.0003. LNP diameter, reported from the four libraries combined, was independent of (D) cholesterol molar ratio and (E) helper lipid molar ratio, average ± SD.

Fig. 2.

The incorporation of cationic cholesterol into LNPs modulates systemic in vivo mRNA delivery. (A) Each LNP library composed of LNPs carrying Cre mRNA and a DNA barcode was administered to Ai14 mice through tail vein injection. Three to four days post injection, five organs were isolated and tdTomato expression was quantified in 22 cell types. tdTomato⁺ cell types were sorted using FACS. Barcodes were extracted from the sorted tdTomato⁺ cell types and sequenced using NGS. NGS quantifies LNP in vivo delivery at cell-type level. (B) Normalized delivery of LNPs in each library across all cell types. Naked barcodes, which were not encapsulated in LNPs, were found less than barcodes carried by LNPs. Normalized deliveries were calculated through a two-step process. First, the count of each barcode within a cell type of interest was divided by the total counts of all barcodes in that same cell type. Second, the barcode count was normalized to the LNP input sample (LNP pool injected into mice). (C) LNPs containing cationic cholesterols showed lower lung:liver mRNA delivery than LNPs with cationic helper lipids. LNPs composed of cationic cholesterols and cationic helper lipids improved nonliver:liver mRNA delivery. One-way ANOVA, ****P < 0.0001, ***P = 0.0007, **P < 0.006, *P < 0.04, average ± SD. (D) (+ 0) library targeted all liver cell types as opposed to (0 +). Unpaired t test, ****P < 0.0001, ***P = 0.0005, **P = 0.006, average ± SD.

Fig. 3.

Location of positive charge impacts systemic in vivo nonliver:liver mRNA delivery. (A) Top-performing LNPs from (0 +) and (+ 0) screens carrying the same mole ratios of four components. (B) These LNPs formed small and monodisperse particles and carried similar positive charge. (C) LNP⁺⁰ outcompetes LNP⁰⁺ in liver delivery in all cell types. mRNA delivery was also quantified in (D) lung, (E) kidney, (F) heart, and (G) spleen. (D–F) LNP⁺⁰ and LNP⁰⁺ led to a significant differential mRNA delivery to lung ECs, lung immune cells, kidney immune cells, and heart ECs. Unpaired t test, ****P < 0.0001, ***P = 0.0009, **P < 0.0095, *P < 0.045, average ± SD.

Fig. 4.

LNP⁺⁺ delivers mRNA to heart ECs as well as lung cell types. (A) LNP⁺⁺ was composed of DC-cholesterol and DOTAP as cationic cholesterol and cationic helper lipid components, respectively. (B and C) LNP⁺⁺ improved lung:liver mRNA delivery and led to significant heart EC delivery. Average ± SD. (D) LNP⁺⁺ targeted lung and heart imaged at single-cell level using HR-FLFM. (E) RNAscope imaging of lung, heart, and liver. Scale bar on the image: 50 µm. (F) t-SNE plot representation of heart cells isolated from mice injected with either PBS or LNP⁺⁺. (G) tdTomato mRNA expression was overlaid on heart cells. LNP⁺⁺ predominantly delivered mRNA to heart ECs confirmed at single-cell level via scRNA-seq.

Fig. 5.

LNP⁺⁺ mediates systemic lung epithelial and stem-like cell mRNA delivery. (A) LNP⁺⁺ carrying aVHH mRNA was injected to BL/6 mice at a dose of 1.5 mg/kg. One day postinjection, aVHH expression was measured in lung, heart, kidney, liver, and spleen. (B) mRNA delivery to lung non-EC cell types, which are less physically accessible through blood vessels, was explored. (C) LNP⁺⁺ led to mRNA delivery to epithelial cells, stem-like cells, and immune cells. Unpaired t test, ****P < 0.0001, ***P = 0.0005, *P < 0.048, average ± SD.