α-bisabolol-loaded lipid-core nanocapsules reduce lipopolysaccharide-induced pulmonary inflammation in mice

Abstract

Acute respiratory distress syndrome (ARDS) is a severe clinical condition of respiratory failure due to an intense inflammatory response with different etiologies. Despite all efforts, therapy remains limited, and ARDS is still associated with high mortality and morbidity. Plants can provide a vast source of active natural products for the discovery of new drugs. α-bisabolol (α-bis), a constituent of the essential oil from chamomile, has elicited pharmacological interest. However, the molecule has some limitations to its biological application. This study was conducted to develop a drug delivery system using lipid-core nanocapsules (LNCs) to improve the anti-inflammatory effects of orally administered α-bis. α-bis-loaded LNCs (α-bis-LNCs) were prepared by interfacial deposition of poly(ε-caprolactone) and orally administered in a mouse model of ARDS triggered by an intranasal administration of lipopolysaccharide (LPS). We found that α-bis-LNCs (30, 50, and 100 mg kg^-1) significantly reduced airway hyperreactivity (AHR), neutrophil infiltration, myeloperoxidase activity, chemokine levels (KC and MIP-2), and tissue lung injury 18 hours after the LPS challenge. By contrast, free α-bis failed to modify AHR and neutrophil accumulation in the bronchoalveolar lavage effluent and lung parenchyma and inhibited elevation in the myeloperoxidase and MIP-2 levels only at the highest dose. Furthermore, only α-bis-LNCs reduced LPS-induced changes in mitogen-activated protein kinase signaling, as observed by a significant reduction in phosphorylation levels of ERK1/2, JNK, and p38 proteins. Taken together, our results clearly show that by using LNCs, α-bis was able to decrease LPS-induced inflammation. These findings may be explained by the robust increase of α-bis concentration in the lung tissue that was achieved by the LNCs. Altogether, these results indicate that α-bis-LNCs should further be investigated as a potential alternative for the treatment of ARDS.

Keywords: acute respiratory distress syndrome; anti-inflammatory effects; drug delivery; nanotechnology; pulmonary inflammation; α-bisabolol.

Figure 1

Effect of treatment with α-bis-LNCs in pulmonary elastance and airway hyperresponsiveness induced by LPS. Notes: The animals were pretreated orally with α-bis, α-bis-LNCs (30, 50, or 100 mg kg⁻¹), or ULNCs. After 4 hours, the mice were anesthetized and challenged intranasally with LPS (25 μg/25 μL) or vehicle. Airway responsiveness was measured by changes in airway elastance after aerosolization of methacholine 18 hours after LPS instillation. Data are expressed as the mean ± SEM (n=5–7). ⁺P<0.05 compared with the saline group; *P<0.05 compared with the LPS-induced group; ^§P<0.05 compared with respective α-bis doses. Abbreviations: α-bis, α-bisabolol; α-bis-LNCs, α-bisabolol-loaded lipid-core nanocapsules; LNCs, lipid-core nanocapsules; ULNCs, drug-unloaded nanocapsules; LPS, lipopolysaccharide; SEM, standard error of mean.

Figure 2

Effect of treatment with α-bis-LNCs on migration of neutrophils in bronchoalveolar lavage fluid. Notes: The mice were treated with α-bis, α-bis-LNCs (30, 50, or 100 mg kg⁻¹), or ULNCs 4 hours before LPS or saline instillation. The bronchoalveolar lavage was performed with PBS + EDTA 18 hours later to collect cells. Then, the lungs were perfused and collected to measure MPO activity. (A) Neutrophils and (B) myeloperoxidase activity. Data are expressed as the mean ± SEM (n=5–7). ⁺⁺⁺P<0.001 compared with the saline group; **P<0.01 and ***P<0.001 compared with the LPS-induced group; ^§P<0.05 compared with α-bis. Abbreviations: α-bis, α-bisabolol; α-bis-LNCs, α-bisabolol-loaded lipid-core nanocapsules; LNCs, lipid-core nanocapsules; BALF, bronchoalveolar lavage fluid; ULNCs, drug-unloaded nanocapsules; LPS, lipopolysaccharide; MPO, myeloperoxidase; SEM, standard error of mean.

Figure 3

Effect of treatment with α-bis-LNCs on chemokine levels. Notes: The mice were treated with α-bis, α-bis-LNCs (30, 50, or 100 mg kg⁻¹), or ULNCs 4 hours before LPS or saline instillation. The lungs were perfused and collected to measure levels of (A) KC and (B) MIP-2 by ELISA. Data are expressed as the mean ± SEM (n=5–7). ⁺⁺P<0.01 and ⁺⁺⁺P<0.001 compared with the saline group; **P<0.01 compared with the LPS-induced group; *P<0.05 compared with the LPS-treated group; ^§P<0.05 compared with α-bis. Abbreviations: α-bis, α-bisabolol; α-bis-LNCs, α-bisabolol-loaded lipid-core nanocapsules; LNCs, lipid-core nanocapsules; ELISA, enzyme-linked immunosorbent assay; ULNCs, drug-unloaded nanocapsules; LPS, lipopolysaccharide; SEM, standard error of mean.

Figure 4

Effect of treatment with α-bis-LNCs on tissue damage. Notes: The mice were treated with α-bis, α-bis-LNCs (30, 50, or 100 mg kg⁻¹), or ULNCs 4 hours before LPS or saline instillation. The lungs were perfused and collected in formalin. Lung serial sections were embedded in paraffin by routine methods and stained with H&E. (A) Microscopic analysis of stained tissue sections: a, saline; b, LPS; c, LPS + ULNCs; d, α-bis 30 mg kg⁻¹; e, α-bis 50 mg kg⁻¹, f, α-bis 100 mg kg⁻¹; g, α-bis-LNC 30 mg kg⁻¹; h, α-bis-LNC 50 mg kg⁻¹; i, α-bis-LNC 100 mg kg⁻¹; 10× magnification. (B) Histological analysis of lung inflammation by a scoring system. Data are expressed as the mean ± SEM (n=5–7). ⁺P<0.05 compared with the saline group; **P<0.01, and ***P<0.001 compared with the LPS-induced group; ^§P<0.05 compared with α-bis. Abbreviations: α-bis, α-bisabolol; α-bis-LNCs, α-bisabolol-loaded lipid-core nanocapsules; LNCs, lipid-core nanocapsules; ULNCs, drug-unloaded nanocapsules; LPS, lipopolysaccharide; SEM, standard error of mean.

Figure 5

Effect of treatment with α-bis-LNCs on MAPK pathway activation. Notes: The lungs were perfused and collected to analyze protein expression 18 hours after LPS challenge and the pretreatment with α-bis, α-bis-LNCs, or ULNCs at a dose of 50 mg kg⁻¹. Representative Western blotting analysis of the phosphorylation state of (A) p38, (B) ERK1/2, and (C) JNK. The densitometric values obtained for phosphorylated and total protein from treatments were normalized to their respective controls that were not exposed to LPS (control bar; 100%). Data are expressed as a ratio of the normalized percentages of phosphorylated and total protein. Bars represent the mean ± SEM (n=5–7). ⁺P<0.05 compared with the saline group; *P<0.05, **P<0.01, and ***P<0.001 compared with the LPS-induced group. Abbreviations: α-bis, α-bisabolol; α-bis-LNCs, α-bisabolol-loaded lipid-core nanocapsules; LNCs, lipid-core nanocapsules; ULNCs, drug-unloaded nanocapsules; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; SEM, standard error of mean.

Figure 6

Quantification of α-bis in lung tissue. Notes: The amount of pulmonary α-bis was analyzed by using high-performance liquid chromatography in mice treated with α-bis or α-bis LNCs (30, 50, or 100 mg kg⁻¹) and challenged with LPS. The α-bis amount was expressed by μg of α-bis per gram of lung tissue. Data are expressed as the mean ± SEM (n=5–7). ^§P<0.05 compared with the respective doses of α-bis. Abbreviations: α-bis, α-bisabolol; α-bis-LNCs, α-bisabolol-loaded lipid-core nanocapsules; LNCs, lipid-core nanocapsules; LPS, lipopolysaccharide; SEM, standard error of mean.