Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation

Abstract

Mucoadhesive particles (MAP) have been widely explored for pulmonary drug delivery because of their perceived benefits in improving particle residence in the lungs. However, retention of particles adhesively trapped in airway mucus may be limited by physiologic mucus clearance mechanisms. In contrast, particles that avoid mucoadhesion and have diameters smaller than mucus mesh spacings rapidly penetrate mucus layers [mucus-penetrating particles (MPP)], which we hypothesized would provide prolonged lung retention compared to MAP. We compared in vivo behaviors of variously sized, polystyrene-based MAP and MPP in the lungs following inhalation. MAP, regardless of particle size, were aggregated and poorly distributed throughout the airways, leading to rapid clearance from the lungs. Conversely, MPP as large as 300 nm exhibited uniform distribution and markedly enhanced retention compared to size-matched MAP. On the basis of these findings, we formulated biodegradable MPP (b-MPP) with an average diameter of <300 nm and examined their behavior following inhalation relative to similarly sized biodegradable MAP (b-MAP). Although b-MPP diffused rapidly through human airway mucus ex vivo, b-MAP did not. Rapid b-MPP movements in mucus ex vivo correlated to a more uniform distribution within the airways and enhanced lung retention time as compared to b-MAP. Furthermore, inhalation of b-MPP loaded with dexamethasone sodium phosphate (DP) significantly reduced inflammation in a mouse model of acute lung inflammation compared to both carrier-free DP and DP-loaded MAP. These studies provide a careful head-to-head comparison of MAP versus MPP following inhalation and challenge a long-standing dogma that favored the use of MAP for pulmonary drug delivery.

Keywords: biodegradable nanoparticles; controlled release; inhaled drug delivery; lung inflammation; mucociliary clearance; mucus-penetrating particles.

Fig. 1. Characterization of GRAS-based biodegradable NP.

Ex vivo stability of NP in BALF at room temperature for 2 or 24 hours, as measured by (A) hydrodynamic diameter and (B) PDI. (C) Transmission electron micrographs of DP/PLGA/F127 and PLGA/F68 NP. Scale bars, 100 nm. (D) Release kinetics of DP from DP/PLGA/F127 and DP/PLGA/F68 NP. Data represent means ± SD. *P < 0.05, **P < 0.01.

Fig. 2. The diffusion of biodegradable NP in human CF sputum.

MPT was performed on NP administered ex vivo in freshly expectorated CF sputum. (A) Representative trajectories of particles. Scale bar, 1 μm. (B) Median MSD at a time scale of 1 s (n = 4). Data represent means ± SEM. *P < 0.05.

Fig. 3. The distribution and retention of model PS-based NP in the mouse lungs.

Airway distribution of (A) 60-, (B) 100-, (C) 300-, and (D) 1000-nm PS and PS-PEG NP at 30 min after administration. PS (green) and PS-PEG (red) NP were coadministered except for 100-nm NP, which were independently dosed to different mice. Cell nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI). The white and yellow arrows indicate PS NP aggregated in the mucus blanket and PS-PEG NP near the surface of epithelium, respectively. Unlike smaller PS-PEG, 1000-nm PS-PEG sparsely distributed throughout the airway similar to PS (red arrows). The retention of PS (white bars) and PS-PEG (gray bars) NP over time is reported as a percentage of the initial deposited dose for (E) 60-, (F) 100-, (G) 300-, and (H) 1000-nm NP (n > 5). Data represent means ± SEM. **P < 0.01.

Fig. 4. The distribution and retention of PLGA-based biodegradable NP in the mouse lung.

Bronchial distribution of (A) PLGA (green) and PLGA-PEG (red) NP and (B) PLGA/F68 (left panel) and PLGA/F127 (right panel) NP at 30 min after administration. Cell nuclei were stained blue with DAPI. The white and yellow arrows indicate PLGA NP aggregated in the mucus blanket and PLGA-PEG NP near the epithelial surface, respectively. (C and D) Tracheal distribution of PLGA (green) and PLGA-PEG (red) NP at 30 min after administration. The yellow dashed box in (C) highlights a submucosal gland covered with PLGA-PEG NP. The white arrows in (C) indicate PLGA NP stuck in the mucus blanket. The same gland can be observed at higher magnification in (D) where the yellow arrow points out deep penetration of PLGA-PEG NP into the gland. (E) The retention of PLGA (white bars) and PLGA-PEG (gray bars) NP over time in BALF. (F) The retention of PLGA and PLGA-PEG NP in the entire lung over time (n = 5). Representative images for PLGA and PLGA-PEG NP exposed lungs over time are shown. The percentage of retention is reported as the percentage of the initial fluorescence (FL) for PLGA (open squares, solid line) and PLGA-PEG (open circles, dotted line) NP. Data represent means ± SEM. *P < 0.05, **P < 0.01.

Fig. 5. In vivo anti-inflammatory effects of GRAS-based biodegradable NP carrying DP in the lungs of mice challenged with Pseudomonas aeruginosa LPS.

Mice were challenged twice with LPS at 0 and 6 hours. At t = 24 hours, LPS-treated mice received DP/PLGA/F127 or DP/PLGA/F68 NP at a dose of 1 mg/kg. Control LPS-treated mice received either carrier-free DP or saline. Mice were sacrificed at 48 hours for BALF analysis. (A) Total inflammatory cell counts. (B) Concentration of TNF-α. Data represent means ± SD. *P < 0.05, **P < 0.01.