Lung targeted liposomes for treating ARDS

Abstract

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1β cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.

Keywords: ARDS; COPD; Covid-19; Liposome; Lung inflammation; Mucus; Nanotechnology; Pulmonary.

Graphical abstract

Fig. 1

Physiochemical characterization of liposomes loaded with MPS and NAC. A) Liposomes loaded with two medicines (methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucus active agent) were synthesized using a three-step formulation process. B) To reach high loading levels of both drugs and a stable formulation process we followed the following optimization methodology. Optimization steps varied drug concentrations, temperature, drug loading time, intra- and extra-liposomal pH, and the Active Loading (AL) salt concentrations. Selected conditions are labeled in yellow. Unfavorable outcomes are labeled in grey, and conditions leading to them in white. C) Summary table of final DPPC liposomal drugs formation process conditions and drugs' encapsulation analysis. (B—C) Drug and lipid concentrations were analyzed using HPLC-ELSD. Particle concentration and size were measured using dynamic light scattering (DLS). D) Schematic of DPPC liposomal drug formulation loaded with MPS and NAC. Formulation includes (dipalmitoyl-phosphatidylcholine) DPPC:cholesterol:DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-polyethylene glycol-2000) (60:35:5 M ratio), with MPS and NAC encapsulated in the liposomal core. E) Cryo-TEM images of the MPS and NAC drug-loaded DPPC liposomes (scale bar = 100 nm), MPS liposomes (F) and NAC liposomes (G). H—I) Liposome size distribution and zeta potential measurement using DLS. J) In vitro Release of MPS and NAC from DPPC liposomes (n = 3). Active Loading (AL); Methylprednisolone succinate (MPS); n-acetyl cysteine (NAC). Fig. 1D was created using Biorender.com. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

NAC increases mucus permeability and MPS reduces macrophage cytokine secretion. A-B) Measuring of LPS-induced macrophage cytokine (TNFα and nitric oxide (NO)) levels following liposomes treatments. One-way ANOVA and one-tailed t-test were performed using PRISM: ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001. Asterisk above bars connecting columns represents a significant difference between groups. C) Confocal images of 100 nm liposomes of NAC treated, or untreated, mucus's penetration. D) In-vitro mucosal barrier permeation assay – cells were plated on 24 walls petri dish and a layer of 200 μL synthetic mucus was applied on top of the cells as a biological barrier. The ability of nanoparticles to cross the mucus layer and be taken up by the underlying cells was quantified over time using an automated microscope setup. E) Automated microscopy images of Caco-2 cellular uptake of Cy-7 fluorescently labeled 100 nm liposomes. The uptake is presented as mean ± SD. One-way ANOVA with an adjusted P-value of multiple comparisons tests was used for statistical analysis; ^⁎⁎P < 0.01; ^⁎P < 0.05. Fig. 2c and 2D was created using Biorender.com.

Fig. 3

In vivo lung inflammation model. A) LPS is administrated endotracheally to induce inflammation. Six hours later, intravenous (IV) or endotracheal (ET) or both IV plus ET treatments were initiated. B) H&E staining of healthy and inflamed lungs 24 h after LPS administration: Arrows indicate, A- distal airway, B- blood vessel, C- alveolar ducts, D- type 1 pneumocytes epithelial cells; E- type 2 pneumocytes epithelial cells, F- alveolar macrophages, G-endothelial blood vessel cell. H- neutrophils and macrophages within airspaces (scale bar = 200 μm and in zoom-in image scale bar is 20 μm) C) Analysis of Inflammatory markers kinetics. TNFα, IL1α, IL1β and IL6, levels in BAL fluid were quantified using ELISA assay. Healthy mice (n = 1), LPS mice (n = 3), Dunnett's multiple comparison test ^⁎⁎P < 0.01; ^⁎⁎P < 0.001; ^⁎⁎⁎P < 0.0001. Asterisk above bars connecting column represents significant difference between groups. Fig. 3A was created using Biorender.com.

Fig. 4

Biodistribution of liposomes in inflamed lung. A) Ex-vivo representative images of healthy and inflamed lungs at varying time points after IV injection of rhodamine and Gd labeled liposomes taken by whole animal IVIS imaging system. B) Biodistribution of rhodamine and Gd labeled liposomes at different time points from IV injection as measured using elemental analysis (ICP-OES) of the lungs, heart, liver, spleen, kidneys and blood. C) Ex-vivo representative IVIS images of lungs at varying time points after ET administration of rhodamine and Gd-labeled liposomes. D) Biodistribution rhodamine and Gd labeled liposomes at different time points from ET administration as measured by ICP-OES in lungs, heart, liver, spleen, kidneys and blood. Healthy mice (n = 4), LPS mice (n = 3). One way ANOVA and one tailed t-test were performed using PRISM: ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001. Asterisk above bars connecting columns represents a significant difference between groups. E) Confocal images of inflamed lung cryo-sections following Cy3-labeled liposomes administered via ET alongside Cy5-labeled liposomes administrated IV. Nuclei were stained using DAPI. Arrows exhibit high intensity of liposomes present. Arrowheads show liposomes in a blood vessel and arrows show liposomes in the airway. Scale bar = 200 μm.

Fig. 5

Efficacy experiments of the DPPC liposomal drug delivered ET, IV or both. A) Representative images of healthy lungs, untreated inflamed lungs, and inflamed lungs after IV plus ET, IV and ET treatment of DPPC liposomal drugs. B) H&E sections of lungs treated IV, ET or IV plus ET, with free drugs, empty DPPC liposomes or drug loaded DPPC liposomes. Scale bar 50 μm (X23). Square shows magnification of X150 exhibiting infiltration of lymphocytes into alveoli. C) Levels of bronchoalveolar lavage (BAL) fluid pro-inflammatory cytokines were quantified after different treatments (ET, IV and IV + ET), TNFα, IL1β, and IL-6 levels in the BAL fluid. One-way ANOVA and one-tailed t-test were performed using PRISM: ^⁎P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001. The asterisk above bars connecting columns represents a significant difference between groups.