(T1430-01-05) Mechanistic Investigation of Side-Effects of Lung Tropic Lipid Nanoparticles

Purpose: Lipid-nanoparticles (LNPs) can be localized to the lung using physicochemical or antibody-mediated strategies. In this study, we compare the side-effects induced by: 1) LNPs targeted primarily to lung endothelial cells via conjugation with an antibody against platelet endothelial cell adhesion molecule (PECAM), and 2) LNPs formulated with the cationic lipid DOTAP which induces lung tropism. We show that DOTAP LNPs activate coagulation in naive mice and this effect is exacerbated in an animal model of acute lung injury achieved by pre-treatment with nebulized LPS. Mechanistic studies reveal that this thrombotic toxicity is associated with the binding of fibrinogen to DOTAP LNPs as part of the protein corona and this effect is ameliorated by pre-treatment with the anticoagulant heparin.

Methods: Platelet count was measured in mice using a Hematology Analyzer while plasma thrombin-antithrombin levels were measured via ELISA. To measure the in vitro binding of fibrinogen to LNPs, fibrinogen was first labeled with an Alexa Fluor 488 fluorophore. LNPs were incubated in plasma doped with fluorescently labeled fibrinogen such that the total fibrinogen concentration was 3mg/ml (physiological value) and incubated for 10 minutes. Nanoparticle Tracking Analysis was carried out with the software set with a 488nm filter to detect only fluorescent fibrinogen. To determine the in vivo biodistribution of fibrinogen, radioactively labeled fibrinogen was injected into mice. LNPs were injected two minutes after and allowed to circulate for 30 minutes. The mice were sacrificed and the organs and blood were harvested and the radioactivity in each organ was measured with a gamma counter. For analysis of luciferase expression, each organ was homogenized after which a luciferin substrate was added to a solution of the organ homogenate and luminescence was quantified with a luminometer.

Results: We show that DOTAP LNPs induce coagulation in naive mice and this effect is exacerbated in nebulized-LPS treated mice. DOTAP LNPs decrease platelet count >1.3x in naive mice and >2x in nebulized-LPS treated mice. Additionally, DOTAP LNPs increase plasma thrombin-antithrombin levels (a marker of net coagulation) by >2x in naive mice and >2.5x in nebulized-LPS mice. These effects on platelet count and thrombin-antithrombin levels were not observed with PECAM LNPs or control LNPs formulated without DOTAP. To determine the mechanism through which coagulation occurs, we tested the binding of fibrinogen to DOTAP LNPs in vitro using Nanoparticle Tracking Analysis (NTA). We observed that DOTAP LNPs bind to fibrinogen >20X more than PECAM LNPs or control LNPs formulated without DOTAP. In vivo biodistribution of radiolabeled fibrinogen shows that in the presence of DOTAP LNPs, there is a significant increase of fibrinogen localized in the lung. The fibrinogen localization ratio (the ratio of the percentage of injected fibrinogen dose in the lung to that in the blood normalized by organ weights) increased >2.3-fold with DOTAP LNPs and remained unchanged with PECAM-LNPs and control LNPs. Additionally, this effect is ameliorated by pre-treatment with the anticoagulant heparin. Pre-treatment of mice with heparin as an anticoagulant attenuates in vivo luciferase mRNA expression suggesting that coagulation is necessary for the expression of DOTAP LNPs.

Conclusion: In this study, we show that DOTAP LNPs induce coagulation. We demonstrate that the mechanism of this is that DOTAP LNPs bind to fibrinogen, aggregate, and form clots as fibrinogen converts to fibrin and travel to the lungs. Our results also indicate that while coagulation is ameliorated by treatment with heparin, expression is attenuated suggesting that coagulation is necessary for the mRNA expression of DOTAP LNPs.


In vitro Fibrinogen Binding. DOTAP LNPs bind to fibrinogen in vitro >20X more than bare LNPs. Fibrinogen adhesion also causes aggregation of the LNPs likely due to clot formation



In vivo fibrinogen biodistribution. DOTAP LNPs appear to bind to fibrinogen in vivo, aggregate, form clots as fibrinogen converts to fibrin and travel to the lungs. This effect is ameliorated with pre-treatment with heparin.


In vivo Luciferase Expression. Pre-treatment of mice with heparin as an anticoagulant attenuates in vivo luciferase mRNA expression suggesting that coagulation is necessary for expression of DOTAP LNPs