PBAE-PEG-based lipid nanoparticles for lung cell-specific gene delivery

Abstract

Exemplified by successful use in COVID-19 vaccination, delivery of modified mRNA encapsulated in lipid nanoparticles (LNPs) provides a framework for treating various genetic and acquired disorders. However, LNPs that can deliver mRNA into specific lung cell types have not yet been established. Here, we sought to determine whether poly(β-amino ester)s (PBAE) or PEGylated PBAE (PBAE-PEG) in combination with 4A3-SC8/DOPE/cholesterol/DOTAP LNPs could deliver mRNA into different types of lung cells in vivo. PBAE-PEG/LNP was similar to Lipofectamine MessengerMAX followed by PBAE/LNP for mRNA transfection efficiency in HEK293T cells in vitro. PBAE-PEG/LNP administered by intravenous (IV) injection achieved 73% mRNA transfection efficiency into lung endothelial cells, while PBAE-PEG/LNP administered by intratracheal (IT) injection achieved 55% efficiency in lung alveolar type II (ATII) epithelial cells in mice in vivo. PBAE/LNP administered by IT injection were superior for specific delivery into lung airway club epithelial cells compared to PBAE-PEG/LNP. Lipofectamine MessengerMAX was inactive in vivo. 5-Methoxyuridine-modified mRNA was more efficient than unmodified mRNA in vivo but not in vitro. Our findings indicate that PBAE-PEG/LNP and PBAE/LNP can transfect multiple lung cell types in vivo, which can be applied in gene therapy targeting genetic lung diseases.

Keywords: PBAE-PEG; cell-specific; lipid nanoparticle; lung endothelial cells; lung epithelial cells.

Graphical abstract

Figure 1

Creation of PBAE and PEGylated PBAE (A) Uncapped poly(β-amino ester)s (PBAE) polymers were synthesized by the Michael addition reaction of 1,4-butanediol diacrylate and 4-amino-1-butanol to produce the PBAE backbone. Subsequently, uncapped PBAE polymers were capped with 1,3-diaminopropane (capped PBAE). PEGylated PBAE (PBAE-PEG) was synthesized by conjugating methoxy-PEG-succinimidyl succinate to capped PBAE at a 2:1 PEG:PBAE molar ratio. (B) Chemical structure of capped PBAE (PBAE) was characterized by ¹H NMR spectroscopy. (C) Chemical structure of PEGylated PBAE (PBAE-PEG) was characterized by ¹H NMR spectroscopy.

Figure 2

PBAE-PEG or PBAE-based lipid nanoparticles are capable of transfecting mRNA in vitro (A) PBAE-PEG/lipid nanoparticles (LNPs) or PBAE/LNP dissolved in ethanol were assembled by vortex mixing with mRNA dissolved in water at pH 4 and then dialyzed in PBS at pH 7 overnight. (B) Schematic shows PBAE-PEG/LNP (LNP: 4A3-SC8/DOPE/cholesterol/DOTAP) carrying mRNA. (C) Transfection efficiency of PBAE-PEG/LNP or PBAE/LNP at different molar ratios from 0.5:135 to 5:135 (PBAE-PEG:LNP or PBAE:LNP) carrying unmodified luciferase mRNA (2 μg) along with Lipofectamine MessengerMAX was assessed using luciferase assay in HEK293T cells. Forty-eight hours after transfection, luciferase activity was measured. Results are normalized by protein weight and expressed as mean ± SD of replicates for each group (n = 3, unpaired, two-tailed Student’s t test). (D) Transfection efficiency of PBAE-PEG/LNP or PBAE/LNP with unmodified luciferase mRNA at different mass ratios from 10:1 to 90:1 (PBAE-PEG/LNP:mRNA or PBAE/LNP:mRNA) was assessed using luciferase assay in HEK293T cells as described in (C). (E) Hydrodynamic diameters of PBAE-PEG/LNP or PBAE/LNP with unmodified luciferase mRNA at different mass ratios from 10:1 to 90:1 (PBAE-PEG/LNP:mRNA or PBAE/LNP:mRNA) were measured using Zetasizer. Results are expressed as mean ± SD of replicates for each group (n = 3, unpaired, two-tailed Student’s t test). (F) The zeta potential of PBAE-PEG/LNP or PBAE/LNP with unmodified luciferase mRNA at different mass ratios were measured as described in (E). Results are also expressed as described in (E). (G) Diameters of PBAE-PEG/LNP (top) and PBAE/LNP (bottom) with unmodified luciferase mRNA (1 μg/mL) that compose mesoporous structures were 211 and 204 nm, respectively, which were measured by transmission electron microscopy (TEM). (H) The hydrodynamic diameters of PBAE-PEG/LNP carrying unmodified luciferase mRNA at pH 7, 6, and 5 that were measured using a Zetasizer indicate that PBAE-PEG/LNP with unmodified luciferase mRNA were responsive to pH change. Red and blue bars indicate size (nm) and PDI, respectively. Results are expressed as mean ± SD of replicates for each group (n = 3, unpaired, two-tailed Student’s t test). ∗∗∗∗ and ∗∗ indicate p < 0.0001 and p < 0.01, respectively. (I) RNA gel electrophoresis indicates the release of unmodified luciferase mRNA from PBAE-PEG/LNP with unmodified luciferase mRNA at a lower pH (pH 4–6) but not at pH 7. Several panels were created with BioRender.com.

Figure 3

PBAE-PEG/LNP and PBAE/LNP can transfect 5moU mRNA in vitro (A) Transfection efficiency of PBAE-PEG/LNP carrying 5moU or unmodified luciferase mRNA (2 μg) at the mass ratio of 30:1 (PBAE-PEG/LNP:mRNA) was assessed using luciferase assay in HEK293T cells. Forty-eight hours after transfection, luciferase activity was measured. Results are normalized by protein weight and expressed as mean ± SD of replicates for each group (n = 3, unpaired, two-tailed Student’s t test). (B) Fluorescence microscopy was conducted to assess transfection efficiency by PBAE-PEG/LNP or PBAE/LNP carrying 5moU mCherry mRNA (2 μg) using HEK293T cells. Immunofluorescence images were captured 48 h after transfection. Scale bar indicates 275 μm. (C) Transfection efficiency for (B) was quantified by counting fluorescence-positive cells. Results are expressed as mean ± SD of replicates for each group (n = 3, unpaired, two-tailed Student’s t test).

Figure 4

PBAE-PEG or PBAE-based LNPs are capable of transfecting mRNA into lung cells in vivo (A) Different routes of injection (intravenous [IV; top] vs. intratracheal [IT; bottom]) with PBAE-PEG/LNP or PBAE/LNP with 5moU luciferase mRNA were conducted to deliver mRNA to the mouse lung. (The drawing was created with BioRender.com.) (B) Luminescence from whole bodies of NU/J nude mice (n = 4) that were transfected by IV (top) or IT (bottom) injections of PBAE-PEG/LNP with 5moU luciferase mRNA (12.5 μg), PBAE/LNP with 5moU luciferase mRNA (12.5 μg), or PBAE-PEG/LNP with unmodified luciferase mRNA (12.5 μg) were visualized 24 h after transfection using IVIS. The molar ratio of 2.5:135 (PBAE-PEG:LNP or PBAE:LNP) was used. Shown are two representative images of four mice (see Figure S1 for the complete group). (C) Luminescence from different organs (lung, spleen, liver, heart, and kidney) of FVB/N mice (n = 3/group) transfected by IV (top) or IT (bottom) injections of PBAE-PEG/LNP with 5moU or unmodified luciferase mRNA (12.5 μg) was captured 24 h after transfection using IVIS. The molar ratio of 2.5:135 (PBAE-PEG:LNP) was used. Shown are representative images of one mouse (see Figure S2 for three mice at different PBAE-PEG:LNP or PBAE:LNP molar ratios). (D) Luminescence intensity (n = 3/group) that is described in (C) was quantified by ImageJ. Results are expressed as mean ± SD of triplicates for each group (unpaired, two-tailed Student’s t test). (E) Luminescence from lungs and livers of FVB/N mice (n = 3/group) transfected by IV (left) or IT (right) injections of only LNP, PBAE/LNP, or PBAE-PEG/LNP with 5moU luciferase mRNA (10 μg) was captured 12 h after transfection using IVIS. A molar ratio of 2.5:135 (PBAE-PEG:LNP or PBAE:LNP) was used. (F) Luminescence intensity from lungs and livers described in (E) was quantified by ImageJ. Results are expressed as mean ± SD of triplicates for each group (unpaired, two-tailed Student’s t test).

Figure 5

PBAE-PEG/LNP and PBAE/LNP deliver mRNA into lung endothelial cells by IV injection and lung epithelial cells by IT injection (A) Gating strategy of fluorescence-activated cell sorting (FACS) analysis for the identification of cell lineage populations (CD31⁺CD45^– as endothelial cells, CD326⁺CD45^– as epithelial cells, CD140a⁺CD45^– as fibroblasts, and CD45⁺ as immune cells) from whole-lung single-cell suspension is shown. (B) Representative FACS histograms show tdTomato expression in endothelial cells (CD31⁺CD45^–), epithelial cells (CD326⁺CD45^–), lung fibroblasts (CD140a⁺CD45^–), and immune cells (CD45⁺) from lungs of Ai14 tdTomato mice 4 days after IV or IT injection of PBAE-PEG/LNP and PBAE/LNP carrying 5moU Cre mRNA (12.5 μg administered twice on days 0 and 2). (C) Graph displays the frequency of tdTomato^high cells in endothelial cells, epithelial cells, fibroblasts, and immune cells obtained as described in (B). Results were pooled from three independent experiments with four mice per group. Each symbol represents an individual mouse. Results are expressed as mean ± SD of replicates for each group (n = 4, unpaired, two-tailed Student’s t test). ∗∗∗∗, ∗∗∗, ∗∗, and ∗ indicate p < 0.0001, p < 0.001, p < 0.01, and p < 0.05, respectively.

Figure 6

IV injection of PBAE-PEG/LNP and PBAE/LNP carrying mRNA targets ERG⁺ lung endothelial cells Lungs of Ai14 tdTomato mice 4 days after transfection with PBAE-PEG/LNP and PBAE/LNP carrying 5moU Cre mRNA were harvested and sectioned for immunofluorescence staining to identify which lung cell types were transfected with Cre mRNA (12.5 μg administered twice on days 0 and 2), resulting in tdTomato expression as described in Figure 5. Cell nuclei were counterstained with DAPI (blue). Shown are representative images from three mice. Scale bar indicates 100 μm. (A) tdTomato (red) was co-expressed with the endothelial cell marker ERG (green). (B) tdTomato (red) was co-expressed with neither the lung epithelial cell marker NKX2-1 (white) nor the alveolar type II (ATII) epithelial cell marker LAMP3 (green). (C) tdTomato (red) was co-expressed with neither the airway club epithelial cell marker SCGB1A1 (white) nor the airway ciliated epithelial cell marker TUBA1A (green). (D) tdTomato (red) was not co-expressed with the fibroblast marker MEOX2 (green).

Figure 7

IT injection of PBAE-PEG/LNP with mRNA targets both NKX2-1/LAMP3⁺ ATII epithelial cells and SCGB1A1⁺ airway club epithelial cells, while PBAE/LNP with mRNA targets SCGB1A1⁺ airway club epithelial cells better than NKX2-1/LAMP3⁺ ATII epithelial cells in mouse lungs Lungs of Ai14 tdTomato mice 4 days after transfection with PBAE-PEG/LNP and PBAE/LNP carrying 5moU Cre mRNA were harvested and sectioned for immunofluorescence staining to identify which lung cell types were transfected with Cre mRNA (12.5 μg administered twice on days 0 and 2), resulting in tdTomato expression as described in Figure 5. Cell nuclei were counterstained with DAPI (blue). Shown are representative images from three mice. Scale bar indicates 100 μm. (A) tdTomato (red) was not co-expressed with the endothelial cell marker ERG (green). (B) tdTomato (red) was co-expressed with the lung epithelial cell marker NKX2-1 (white) and the ATII epithelial cell marker LAMP3 (green). (C) tdTomato (red) was co-expressed with the airway club epithelial cell marker SCGB1A1 (white) but not the airway ciliated epithelial cell marker TUBA1A (green). (D) tdTomato (red) was not co-expressed with the fibroblast marker MEOX2 (green).