Macrophage-Targeted Lipid Nanoparticle Delivery of microRNA-146a to Mitigate Hemorrhagic Shock-Induced Acute Respiratory Distress Syndrome

Abstract

The pro-inflammatory response of alveolar macrophages to injurious physical forces during mechanical ventilation is regulated by the anti-inflammatory microRNA, miR-146a. Increasing miR-146a expression to supraphysiologic levels using untargeted lipid nanoparticles reduces ventilator-induced lung injury but requires a high initial dose of miR-146a making it less clinically applicable. In this study, we developed mannosylated lipid nanoparticles that can effectively mitigate lung injury at the initiation of mechanical ventilation with lower doses of miR-146a. We used a physiologically relevant humanized in vitro coculture system to evaluate the cell-specific targeting efficiency of the mannosylated lipid nanoparticle. We discovered that mannosylated lipid nanoparticles preferentially deliver miR-146a to alveolar macrophages and reduce force-induced inflammation in vitro. Our in vivo study using a clinically relevant mouse model of hemorrhagic shock-induced acute respiratory distress syndrome demonstrated that delivery of a low dose of miR-146a (0.1 nmol) using mannosylated lipid nanoparticles dramatically increases miR-146a levels in mouse alveolar macrophages and decreases lung inflammation. These data suggest that mannosylated lipid nanoparticles may have the therapeutic potential to mitigate lung injury during mechanical ventilation.

Keywords: Mannosylated lipid nanoparticle; acute respiratory distress syndrome; alveolar macrophages; inflammation; microRNA.

Figure 1.. Fabrication and characterization of mannosylated lipid nanoparticle (MLNP).

a) Schematic of MLNP fabrication. Created with BioRender.com. b) Representative cryo-TEM image of miR-146a loaded MLNPs. Scale bar: 100nm. The experiment was performed twice. c) Representative size distribution of empty MLNPs and miR-146a loaded MLNPs. The experiment was performed three times. d) Storage stability in terms of size/diameter (left y-axis, red line) and polydispersity index (PDI) (right y-axis, black line) of empty MLNPs and miR-146a loaded MLNPs determined by dynamic light scattering (DLS) at time intervals of 0, 3, 7, and 14 days.

Figure 2.. Cellular uptake of miR-146a loaded MLNPs in primary alveolar macrophages.

a) Transfection efficiency of miR-146a in primary alveolar macrophages (AMs) obtained from donor 7 following delivery of miR-146a with different lipid nanoparticle formulations LNP, 2-MLNP, 4.85-MLNP and 9.3-MLNP (LNP=non-mannosylated lipid nanoparticles, MLNP=mannosylated LNPs containing 2%, 4.85% or 9.3% of mannose-conjugated PA-PEG lipid respectively), calculated by ΔΔCt method, normalized to scramble-loaded LNP controls. Data are normally distributed by Shapiro-Wilk test. Statistical analysis was performed via one-way ANOVA with Tukey’s multiple comparisons test. b) miR-146a levels in AMs following 9.3-MLNP delivery of 5nM, 50nM or 100nM miR-146a, compared to scr-loaded 9.3-MLNP. Data are normally distributed by Shapiro-Wilk test. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. c) miR-146a levels in AMs following delivery of miR-146a using LNPs or 9.3-MLNPs in the presence or absence of 20 mM mannose. Data were analyzed by Kruskal-Wallis with Dunn’s multiple comparisons test. Statistical differences are denoted as *p<0.05, **p<0.005, ***p<0.001. Data are presented as Min to Max. n=3 wells per group. The experiment was performed twice.

Figure 3.. MLNPs do not induce toxicity on cells in vitro and on mice in vivo.

Cell viability of a) AMs and b) pneumocytes after 24 h exposure to empty MLNP with various lipid concentrations by MTS assay. Cells with media alone were used as controls. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. **p < 0.005, ****p < 0.0001. Data are shown as means ± SEM, n=3 wells per group. c) The amount of total protein in bronchoalveolar lavage (BAL) fluid at 2 h after pulmonary administration of empty 9.3-MLNPs, untreated and saline-treated C57BL/6 mice were used as controls, n=3 per group. Effects of treatment of mice with saline or empty 9.3-MLNPs compared to untreated controls (n=3 per group) on d) blood oxygen saturation measured by pulse oximetry 2 h post-administration; e) lung tissue elastance; and f) airway resistance measurements. g) BAL differential cell counts from MLNP treated mice and controls. Data are normally distributed by Shapiro-Wilk test. Data are not significant via one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as Min to Max.

Figure 4.. Mannosylated lipid nanoparticles preferentially deliver miR-146a to primary alveolar macrophages co-cultured with alveolar epithelial cells.

miR-146a levels following LNP or 9.3-MLNP delivery of miR-146a or scramble in flow-sorted pneumocytes and AMs from a) donor 1; b) donor 2; c) donor 3, n=3 wells per group. Data are normally distributed by Shapiro-Wilk test. Statistical analysis was performed separately on LNP data (left panel) and 9.3-MLNP data (right panel) by one-way ANOVA with Tukey’s multiple comparisons test. Statistical differences are denoted as *p<0.05, **p<0.01, ****p<0.0001. Data are presented as Min to Max.

Figure 5.. MLNP delivery of miR-146a more potently dampens pressure-induced inflammation in co-culture.

MIP-1 alpha (MIP-1α), IL8, and IL6 levels were determined following treatment with miR-146a loaded LNPs or 9.3-MLNPs in co-culture of pneumocytes and alveolar macrophages (AMs) from three donor lungs (donor 3, 4, 5) subjected to 16 h of oscillatory pressure at the air-liquid interface, normalized to unpressurized scramble-loaded MLNP treated controls. n = 3 for each donor. a) Fold change in (MIP-1α) secretion; Data are normally distributed by Shapiro-Wilk test. b) Fold change in IL8 secretion; Data are log-normally distributed by Shapiro-Wilk test. C) Fold change in IL6 secretion; Data are log-normally distributed by Shapiro-Wilk test. All Data were analyzed via one-way ANOVA with Sidak’s multiple comparisons test. Statistical differences are denoted as *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. Data are presented as Min to Max.

Figure 6.. In vivo lung cell-distribution of Cy3-Scramble loaded MLNP.

a) Representative fluorescence and DIC images from lung tissue of spontaneously breathing mice treated with Cy3-scramble-miR loaded LNPs or b) Cy3-scramble-miR loaded 9.3-MLNPs by immunofluorescence staining for CD68 (green). Scale bar: 100 μm, n=3 mice per group. c) Percentage macrophage cell area containing Cy3-scramble loaded nanoparticles using immunofluorescence images. **p=0.0055. d) Percentage of the number of macrophages containing Cy3-scramble loaded nanoparticles. **p=0.0013. n=19 high-power fields from one animal per group and n=15 high-power fields from the other two animals per group. Data were analyzed by Mann-Whitney test. Data are presented as Min to Max.

Figure 7.. MLNP delivery of miR-146a mitigates lung injury in a model of hemorrhagic shock-induced ARDS.

a) miR-146a levels in RNA from BAL cell pellets following nanoparticle delivery in HS-ARDS mice. Relative expression determined by ΔΔCt method, normalized to scramble control. ****p<0.0001. b) TRAF6 mRNA levels from BAL cells by ΔΔCt method normalized to scramble. c) BAL IL6 level from miR-146a or scramble (scr) loaded nanoparticles treated HS-ARDS mice. *p<0.05, **p<0.005. d) BAL IL8 level from miR-146a or scramble (scr) loaded nanoparticles treated HS-ARDS mice. **p<0.01. e) BAL protein concentration from miR-146a and scr nanoparticles treated HS-ARDS mice. f) Change in lung elastance during 2 h period of ventilation following treatment with miR-146a or scr loaded nanoparticles. g) Oxygenation throughout duration of ventilation measured via pulse oximetry. Data were analyzed via repeated measures two-way ANOVA with Tukey’s multiple comparison test. h) Percentage macrophages in BAL cells by differential counts after ventilation. For all panels, n=8 for scramble group and n=6 for miR groups. All Data are normally or log-normally distributed by Shapiro-Wilk test. Data were analyzed via one-way ANOVA with Tukey’s multiple comparison test, otherwise stated eleswhere. Data are presented as Min to Max.