(W0930-03-14) Macrophage-Specific Nanoparticle Delivery of MicroRNAs to Mitigate Ventilator-Induced Lung Injury during Acute Respiratory Distress Syndrome (ARDS)

Purpose: The acute respiratory distress syndrome (ARDS) is a life-threatening condition that requires life-support therapy with mechanical ventilation (MV). However, MV generates mechanical forces that can exacerbate lung injury, known as ventilator-induced lung injury (VILI) [1]. These forces can damage the alveolar-capillary barrier and trigger the release of proinflammatory mediators which further damage the barrier. Recently, our laboratory demonstrated that alveolar macrophages (AMs), the resident immune cells of the lung, respond to mechanical forces by upregulating an anti-inflammatory microRNA, miR-146a, but that this native mechanotransduction response is insufficient to mitigate VILI [2]. In addition, we have demonstrated that overexpression of miR-146a in-vivo using untargeted lipid nanoparticles (LNPs) mitigates cytokine release and barrier disruption following injurious ventilation [2]. However, the in-vivo nanoparticle distribution data showed that ~40% of nanoparticles are distributed in non-AMs cells (primarily epithelial cells) [2]. Therefore, in this study we aim to develop mannosylated LNP (M-LNPs) that can deliver miR-146a to only AMs. We hypothesize that this AM-specific delivery of miR-146a can more potently mitigate lung injury and dampen inflammatory signaling pathways during ARDS/VILI.

Methods: LNPs were prepared using an ethanol injection method as previously described [2]. Targeted LNPs were prepared using the same method with an addition of mannose conjugation. Particle size was measured by Dynamic Light Scattering. Nanoparticle encapsulation of pre-miR146a was assessed by gel electrophoresis. To compare the uptake efficiency of mannose-conjugated LNPs (M-LNPs) to that of untargeted LNPs in vitro, primary human alveolar macrophages were transfected with miR-146a loaded untargeted LNPs or targeted M-LNPs. The miR-146a expression in AMs were measured by RT-qPCR. To evaluate if the M-LNPs are AM-specific, primary human epithelial cells (Type I pneumocytes) and primary AMs in a co-culture system were treated with miR-146a loaded M-LNPs. Epithelial cells and AMs were then sorted by a flow cytometer. The miR-146a expression in AMs and epithelial cells were quantified by RT-qPCR. To evaluate the therapeutic potential to mitigate lung injury during mechanical ventilation, we used an in vitro barotrauma model, applied oscillatory pressure to pneumocyte-AM co-culture for 16 hours, and measured release of pro-inflammatory cytokines/chemokines, including interleukin-6 (IL-6), IL-8, and macrophage inflammatory protein-α (MIP-1α).

Results: Targeted M-LNPs were less than 200 nm in size with an encapsulation efficiency of close to 100%. Primary human AMs transfected with 50 nM miR-146a loaded M-LNPs showed ~60-fold higher miR-146a expression compared to that transfected with 50 nM miR-146a loaded untargeted LNPs. Using a pneumocyte-AM co-culture system, we found that M-LNPs preferentially increased miR-146a levels in AMs compared to pneumocytes with p=0.0151 by nonparametric test, while LNP delivered miR-146a to AMs and pneumocytes at similar levels. In response to barotrauma, M-LNPs delivery of miR-146a inhibited IL-8 and MIP-1α release to a greater degree compared to LNP delivery in co-culture.

Conclusion: Targeted M-LNPs preferentially delivered miR-146a to primary AMs in vitro compared to untargeted LNPs resulting in potent inhibition of pressure-induced pro-inflammatory cytokine/chemokine release in co-culture. We are currently investigating the use of targeted M-LNPs for in vivo delivery in murine ARDS model.

References: [1] Carrasco Loza, et al., (2015) Open Respir Med J, 9:112-9 [2] *Bobba, *Fei, et al., (2021) Nat Commun, 12, 289.


Figure 1. M-LNP preferentially increased miR-146a levels in human primary alveolar macrophages (AMs) compared to pneumocytes in co-culture


Figure 2. M-LNP delivery of miR-146a inhibited IL-8 release to a greater degree compared to LNP delivery in co-culture in response to barotrauma.


Figure 3. M-LNP delivery of miR-146a inhibited MIP-1α release to a greater degree compared to LNP delivery in co-culture in response to barotrauma.