Efficient Nebulization and Pulmonary Biodistribution of Polymeric Nanocarriers in an Acute Lung Injury Preclinical Model

Abstract

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by acute hypoxemic respiratory failure. Pneumonia and sepsis are the most common causes, turning ARDS into a critical public health problem. Despite recent advances in pharmacological strategies, clinical trials have not demonstrated a reduction in ARDS-associated mortality. This is in part connected to the singularity of the pulmonary physiological barrier, which hampers drug delivery, specifically at distal areas. To this aim, the use of polymeric nanocarriers as a platform for the efficient delivery of therapeutics to the lungs by nebulization is introduced. Herein, poly(lactic-co-glycolic acid) (PLGA) nanocapsules (NCs) loaded with human serum albumin, as an inhalable nanotherapeutic are prepared. The production of stable NCs aerosols in the inhalable range is achieved using a commercial device, while the nanocarrier's physicochemical parameters are only minimally altered after nebulization. Importantly, in vivo studies with healthy and acute lung injury animals show that after inhalation, the NCs are homogeneously distributed throughout the lungs, arriving at the distal areas. The NCs are internalized by alveolar type II cells, avoiding macrophage-mediated lung clearance. These features make the PLGA NCs excellent vehicles for noninvasive pulmonary delivery, facilitating a ready-to-be-used nanomedicine.

Keywords: inhalation; lung; nanocarriers; poly(lactic‐co‐glycolic acid); vibrating mesh nebulizer.

Figure 1

Aerosol characterization during the nebulization of PLGA/HSA NCs at different concentrations (0.1, 1, and 5 mg mL⁻¹) in water and saline: a) aerosol droplet size (defined as volume median diameter); b) fine particle fraction (defined as the percentage of aerosol with a size below 5 μm); and c) flow rate in mL min⁻¹. Data shown as the mean ± sem (N = 3); *p‐val < 0.05; ***p‐val < 0.001.

Figure 2

Hydrodynamic PLGA/HSA NCs diameter obtained by NTA before and after nebulization at different NC's concentrations (0.1, 1, and 5 mg mL⁻¹) in water and saline; a) comparison of the hydrodynamic diameter mean values; b) 10th, 50th, and 90th percentiles; and c) concentration (error bars are present but hardly visible). Data shown as the mean ± sem (N = 3); ***p‐val < 0.001.

Figure 3

SEM images of PLGA/HSA NCs nebulized in water: a) prenebulization; postnebulization at b) 0.1 mg mL⁻¹, c) 1 mg mL⁻¹, and d) 5 mg mL⁻¹. Scale bar: 2 μm.

Figure 4

Experimental development and evaluation of the ALI animal model: a) in vivo experimental timeline and schematic illustration of the different lung lobes of a rat; b) animal body weight; c) ratio of lung/body weight; and d) infiltration of CD11b+ cells in lung tissue. Data shown as the mean ± sem (n = 2–6); *p‐val < 0.05; **p‐val < 0.001; ***p‐val < 0.001.

Figure 5

In vivo biodistribution of PLGA‐Cy5/HSA NCs in control (healthy) and ALI animals: a) FMI views of the lungs of a control animal, a healthy animal nebulized with NCs, and an ALI animal nebulized with NCs; b) corrected TRE on unilobular lung, superior, middle, and inferior lobes regions of interest (ROIs) of control (healthy) and ALI animals nebulized with NCs; and c) Z‐stacking analysis of lung (unilobular lung, superior, middle, and inferior lobes) tissue slices of animals nebulized with NCs. Scale bar: 20 μm. Green: membranes stained with Cell Mask; red: NCs (Cy5); 60× magnification; average zoom = 1×. Data is shown as the mean ± sem (n = 6).

Figure 6

In vivo cellular uptake of PLGA‐Cy5/HSA NCs in control (healthy) and ALI animals: percentage of a) ATII cells and b) CD11b+ cells from lung tissue homogenates that were positive for Cy5 fluorescence. Healthy (green) and ALI animals (orange) were nebulized with PLGA‐Cy5/HSA NCs and uni‐ and multilobular lungs were analyzed. Data shown as the mean ± sem (n = 6); *p‐val < 0.05.

Figure 7

Scheme of the experimental work: a) commercial nebulizer (Aerogen Solo); b) in vitro nebulization procedure and analytic techniques used for aerosol and nanocarriers characterization; and c) in vivo experimental procedure and techniques used (animals were confined in restraint tubes and spontaneously breathing).