Mechanisms by Which Liposomes Improve Inhaled Drug Delivery for Alveolar Diseases

Abstract

Diseases of the pulmonary alveolus, such as pulmonary fibrosis, are leading causes of morbidity and mortality, but exceedingly few drugs are developed for them. A major reason for this gap is that after inhalation, drugs are quickly whisked away from alveoli due to their high perfusion. To solve this problem, the mechanisms by which nano-scale drug carriers dramatically improve lung pharmacokinetics using an inhalable liposome formulation containing nintedanib, an antifibrotic for pulmonary fibrosis, are studied. Direct instillation of liposomes in murine lung increases nintedanib's total lung delivery over time by 8000-fold and lung half life by tenfold, compared to oral nintedanib. Counterintuitively, it is shown that pulmonary surfactant neither lyses nor aggregates the liposomes. Instead, each lung compartment (alveolar fluid, alveolar leukocytes, and parenchyma) elutes liposomes over 24 h, likely serving as "drug depots." After deposition in the surfactant layer, liposomes are transferred over 3-6 h to alveolar leukocytes (which take up a surprisingly minor 1-5% of total lung dose instilled) in a nonsaturable fashion. Further, all cell layers of the lung parenchyma take up liposomes. These and other mechanisms elucidated here should guide engineering of future inhaled nanomedicine for alveolar diseases.

Keywords: inhaled; nanomedicine; nintedanib; pulmonary fibrosis; surfactant.

Figure 1

Nintedanib‐loaded liposomes have advantageous drug‐loading properties and are stable in ex vivo BALF. a) Schematic of liposome showing phospholipid bilayer with small molecule drug within the aqueous core. b) Negative stain electron microscopy of nintedanib‐loaded liposomes; scale bar, 500 nm. c) Hydrodynamic diameter, PDI, and zeta potential (mV) of empty and NTD‐loaded liposomes. d) Graph adapted from Chemicalize (Copyright 2022 Chemaxon) demonstrates nintedanib species at different pH. Shown in orange at pH 2–6, nintedanib's tertiary amine is protonated; this form is encapsulated in liposomes. Shown in blue at pH ≈9, nintedanib's tertiary amine is in a neutral state. e) Nintedanib leak out of liposomes over 24 h at varied pH shows less leak at neutral pH (as in serum and alveolar surfactant) versus acidic pH (as in endosomes), n = 1 preparation per pH. f) Assessment of fluorescent liposomes in ex vivo BALF up to 24 h, n = 1 per time point. Liposome elution by size exclusion chromatography shows no difference after incubation in BALF, and no free fluorophore peak. g,h) Assessment of fluorescent liposomes in ex vivo BALF up to 24 h, n = 5 technical replicates per time point. g) Size distribution and h) fluorescence intensity by size show complete overlap of all conditions, and therefore no difference after incubation in BALF. Error bars represent SEM.

Figure 2

Inhaled nintedanib‐loaded liposomes confer massive increases in lung half‐life and AUC compared to inhaled or oral free nintedanib. Mice were given one of three nintedanib formulations, shown in a): intratracheal instillation of liposome‐loaded nintedanib (red), intratracheal instillation of free nintedanib (orange), or oral gavage of free nintedanib (blue; noting that clinically the drug is orally administered). Lung and plasma were harvested at time points up to 24 h, and then nintedanib concentration was measured by LC/MS. b,c) Nintedanib concentration over 24 h is shown for each formulation, measured by LC/MS; b) in the lung and c) in plasma. d) AUC and half‐life were calculated using GastroPlus noncompartmental modeling software from data obtained by LC/MS in panels (b) and (c). For oral dosing, AUC is shown for the dose given (60 mg kg⁻¹) and then also normalized to the dose given by intratracheal administration (0.5 mg kg⁻¹). e) Instilled liposomal nintedanib has a 35‐fold and 8000‐fold higher lung AUC compared to oral and instilled free drug (no nanocarrier), respectively. f) Instilled liposomal nintedanib has an eightfold and tenfold increase in lung half‐life compared to free instilled and oral drug, respectively. N = 3, error bars = SEM.

Figure 3

Liposomes remain intact and do not aggregate following intratracheal instillation. Mice were given 10 mg kg⁻¹ lipid of fluorescent liposomes or nintedanib‐loaded fluorescent liposomes via intratracheal instillation, and then BALF was harvested and analyzed by NTA. a) Example primary data showing NTA of endogenous particles in BALF by light scattering (left) and liposomes in BAL by fluorescence scattering (right). b) Normalized histogram of nintedanib‐loaded liposomes in their native form (before being given to mice, dotted green line, fluorescence emission), compared to nintedanib‐loaded liposomes present in BALF 15 min after instillation (solid green line, fluorescence scattering), compared to endogenous particles + nintedanib‐loaded liposomes present in BALF 15 min after instillation (dotted red line). Nintedanib liposomes have the same size distribution before and after lung delivery. c–e) Size distribution of c) BAL‐extracted empty liposomes and e) nintedanib liposomes at 15 min, 4, and 20 h after liposome instillation, each compared to their preinstillation size distribution. d) Average size of liposomes over time [data from histograms in (c) and (e)], noting small size increase with increased time the liposomes have dwelled in the BALF layer of the lungs in vivo. f) Liposomes recovered in BAL over time, as a percentage of initial dose, showing that at least ≈20–30% of liposomes are recoverable intact in BALF as long as 20 h after instillation; inset is the same data depicted as averages. N = 3–6 for empty liposomes; N = 3 for nintedanib liposomes; error bars = SEM.

Figure 4

Inhaled liposomes behavior in BALF, alveolar macrophages, and lung parenchyma: over time, lung parenchyma's share of total lung dose increases; over dose range, macrophages do not demonstrate saturability. Mice were given liposomes labeled with ¹²⁵I radiotracer via intratracheal instillation, then BALF was collected and organs were harvested at various time points as shown. Liposome localization was measured by a gamma counter. a) Schematic demonstrating intratracheal delivery into lungs with division into two initial compartments: the intra‐alveolar compartment (blue), consisting of liposomes taken up by intra‐alveolar leukocytes or suspended in the cell‐free BALF layer; and the parenchymal compartment (pink), which is composed of all lung cells except intra‐alveolar leukocytes. b) Biodistribution over time in blood, lung, stomach, and, in inset graph due to low total levels, in liver, spleen, and intestine. Lipid dose: 2.5 mg kg⁻¹. c–f) Saturability study with varied lipid dose from 1.25 to 30 mg kg⁻¹. Lung compartment biodistribution is divided into c) lung parenchyma, d) total BALF, e) BAL supernatant, and f) BAL pellet. N = 3, error bars = SEM.

Figure 5

Inhaled liposomes home to both alveolar leukocytes and lung parenchymal cells. Mice were given fluorescent liposomes (empty and drug‐loaded) via intratracheal instillation, and then at 20 h whole lung was analyzed. a–f) Prior to harvest and preparation of single‐cell suspension for flow cytometry, BAL and perfusion were performed. a,b) Greater than 75% of alveolar leukocyte populations were associated with liposomes, but only macrophages (B, right panel) demonstrated a large shift in fluorescence, indicating significant uptake. c,d) Comparatively, parenchymal leukocytes (those embedded in the lung parenchyma, or at least firmly attached) were significantly less associated with liposomes (<15%), but parenchymal macrophages still demonstrated a significant shift (D, right panel). e,f) Cells of the lung parenchyma, such as epithelial cells and endothelial cells, were approximately 10% and 35% associated with liposomes, respectively, though with a much smaller shift in fluorescence compared to macrophages. g,h) IHC demonstrates liposomes (red) in all layers of lung tissue in both h) alveolated tissue and g) conducting airways. Airspace leukocytes are seen with significant liposome uptake (arrows). Scale bar = 50 μm. N = 2, error bars = SEM.

Figure 6

Intratracheally delivered liposomes and nintedanib liposomes are nontoxic in vivo. Toxicity studies were performed at 24 h. Naive murine lung is compared to nebulized LPS (a positive control for injury), intratracheal instillation of nintedanib‐loaded liposomes (0.5 mg kg⁻¹ drug and 0.73 mg kg⁻¹ lipid) in sucrose, empty liposomes (2.5 mg kg⁻¹ lipid) in saline, empty liposomes (2.5 mg kg⁻¹ lipid) in sucrose, saline buffer, or sucrose buffer. a) BALF leukocyte count; b) BALF protein concentration; c) weight change from baseline. N = 3 mice per group, one‐way ANOVA; **** p <= 0.0001, ** p <= 0.01. d–g) H&E histology shows no observable toxicity from intratracheal liposomes compared to acid instillation positive control; representative images, n = 3 mice per group with the exception of acid instillation, n = 1 and saline instillation, n = 2.