Oleic acid-based nanosystems for mitigating acute respiratory distress syndrome in mice through neutrophil suppression: how the particulate size affects therapeutic efficiency

Abstract

Background: Oleic acid (OA) is reported to show anti-inflammatory activity toward activated neutrophils. It is also an important material in nanoparticles for increased stability and cellular internalization. We aimed to evaluate the anti-inflammatory activity of injectable OA-based nanoparticles for treating lung injury. Different sizes of nanocarriers were prepared to explore the effect of nanoparticulate size on inflammation inhibition.

Results: The nanoparticles were fabricated with the mean diameters of 105, 153, and 225 nm. The nanocarriers were ingested by isolated human neutrophils during a 5-min period, with the smaller sizes exhibiting greater uptake. The size reduction led to the decrease of cell viability and the intracellular calcium level. The OA-loaded nanosystems dose-dependently suppressed the superoxide anion and elastase produced by the stimulated neutrophils. The inhibition level was comparable for the nanoparticles of different sizes. In the ex vivo biodistribution study, the pulmonary accumulation of nanoparticles increased following the increase of particle size. The nanocarriers were mainly excreted by the liver and bile clearance. Mice were exposed to intratracheal lipopolysaccharide (LPS) to induce acute respiratory distress syndrome (ARDS), like lung damage. The lipid-based nanocarriers mitigated myeloperoxidase (MPO) and cytokines more effectively as compared to OA solution. The larger nanoparticles displayed greater reduction on MPO, TNF-α, and IL-6 than the smaller ones. The histology confirmed the decreased pulmonary neutrophil recruitment and lung-architecture damage after intravenous administration of larger nanoparticles.

Conclusions: Nanoparticulate size, an essential property governing the anti-inflammatory effect and lung-injury therapy, had different effects on activated neutrophil inhibition and in vivo therapeutic efficacy.

Keywords: Acute respiratory distress syndrome; Anti-inflammation; Lipid-based nanoparticles; Neutrophil; Oleic acid; Size.

Fig. 1

The uptake of rhodamine 800-loaded OA nanocarriers by human neutrophils. a The schematic exhibition of the structures of AS, AM, and AL. b Molecular environment (polarity) of the nanocarriers examined by Nile red fluorescence intensity. c The fluorescence intensity of rhodamine 800 in the neutrophils analyzed by flow cytometry. d Confocal microscopy of neutrophils demonstrated that rhodamine 800-loaded OA nanocarriers (red color) were internalized. Neutrophils nucleus stained (blue) were visualized by DAPI. All data represent mean ± SEM (n = 6)

Fig. 2

Neutrophil cytotoxicity assay by treatment of OA-loaded nanocarriers with different droplet sizes. a The neutrophils (6 × 10⁵ cells/ml) were treated with free OA. b The neutrophils (6 × 10⁵ cells/ml) were treated with AS. c The neutrophils (6 × 10⁵ cells/ml) were treated with AM. d The neutrophils (6 × 10⁵ cells/ml) were treated with AL. The cytotoxicity was measured by LDH assay. All data are expressed as the mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to control

Fig. 3

Effects of OA-loaded nanocarriers on superoxide anion release and elastase activity in fMLF-activated human neutrophils (6 × 10⁵ cells/ml). a The measurement of extracellular superoxide production by fMLF/cytochalasin B for 10 min. b Assay of absorbance at 405 nm for continuous measurement of human neutrophil elastase release. All data are expressed as the mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to fMLF-activated group without OA intervention

Fig. 4

The Ca²⁺ mobilization and functional change of fMLF-activated neutrophils. a Fluo-3/AM-labeled neutrophils were treated with free OA and OA-loaded nanocarriers for 5 min. Next, the cells were activated by fMLF. The [Ca²⁺]_i-time curves are shown. b Peak calcium concentration ([Ca²⁺]_i) traces are shown. c Reduction of the time taken to decline to half of its peak values (t_1/2) are shown. All data represent mean ± SEM (n = 6). *p < 0.05, **p < 0.01 as compared to fMLF-activated group without OA intervention

Fig. 5

Effects of OA-loaded nanocarriers on the NET formation. a The PMA-activated neutrophils were treated with free OA. b The PMA-activated neutrophils were treated with AS. c The PMA-activated neutrophils were treated with AM. d The PMA-activated neutrophils were treated with AL. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to PMA-activated group without OA intervention

Fig. 6

Ex vivo bioimaging of organs of the mice receiving intravenous iFlour 790 acid-loaded OA nanocarriers. a The NIR signal of the prepared samples of free iFlour 790 acid and iFlour 790 acid-loaded OA nanocarriers. b The ex vivo bioimaging of organs of representative animals. c The percentage (%) of NIR intensity of iFlour 790 acid in different organs analyzed by Pearl Impulse. The scale of bioimaging was calibrated by the intensity of the formulations for impartial comparison. All data represent mean ± SEM (n = 6). *p < 0.05, **p < 0.01 as compared to AL

Fig. 7

The effect of intravenous free OA and OA-loaded nanocarriers on LPS-induced lung injury in mice. a The lung histology (H&E staining) of LPS-challenged mice treated by free OA and OA-loaded nanocarriers. b The immunohistochemistry (MPO antibody staining) of LPS-challenged mice treated by free OA and OA-loaded nanocarriers. c The immunohistochemistry (Ly6G antibody staining) of LPS-challenged mice treated by free OA and OA-loaded nanocarriers. d MPO expression. e TNF-αexpression. f IL-1β expression. g IL-6 expression. h CXCL-2 expression. All data represent mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to LPS-treated group without OA intervention