Enhancing Lung Recovery: Inhaled Poly(lactic- co-glycolic) Acid Encapsulating FTY720 and Nobiletin for Lipopolysaccharide-Induced Lung Injury, with Advanced Inhalation Tower Technology

Abstract

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), a rapidly progressing respiratory failure condition, results in a high mortality rate, especially in severe cases. Numerous trials have investigated various pharmacotherapy approaches, but their effectiveness remains uncertain. Here, we present an inhaled nanoformulation of fingolimod (FTY720)-nobiletin (NOB)- poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) with good biocompatibility and a sustained-release pharmacological effect. The formulation decreases the toxicity of FTY720 and increases the bioavailability of NOB since we use PLGA with a high biocompatibility to encapsulate FTY720 and NOB at the same time. In vitro, in comparison to treatment with the pure drug, we demonstrated that FTY720-NOB-PLGA NPs can reduce interleukin-6 (IL-6) and reactive oxygen species (ROS) release by macrophages after lipopolysaccharide (LPS) stimulation more efficiently. In vivo, we used an inhalation tower system that allowed the exposure of unanesthetized mice to aerosolized FTY720-NOB-PLGA NPs under controlled conditions. We demonstrated that inhaled FTY720-NOB-PLGA NPs can attenuate lung injury after LPS exposure by suppressing cytokine release, such as IL-6 and tumor necrosis factor-α (TNF-α). The trigger pathway of ALI, including nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and p38 mitogen-activated protein kinase, was also efficiently inhibited. Furthermore, the inhalation treatment provided a good safety profile, without detrimental effects on biochemical markers and lung function. We provided the feasibility of administering inhalation of NPs noninvasively with continuous monitoring of lung function. The aerosolized FTY720-NOB-PLGA NPs we developed show excellent promise for acute lung injury therapy in the future.

Keywords: acute lung injury (ALI); cytokine suppression; fingolimod (FTY720); immune cell infiltration; inhaled nanoformulation; nobiletin (NOB).

Figure 1

Scheme of encapsulating the synergistic effect of the dual drugs in the treatment of ALI.

Figure 2

(A) The number distribution of coencapsulated FTY720 and NOB NPs was prepared by the emulsification method using PLGA. (B) The morphology of the FTY720-NOB-PLGA NPs with a field of view of 30K.

Figure 3

(A) FTIR spectra of the (a) FTY720, (b) NOB, (c) PLGA, and (d) FTY720-NOB-PLGA NPs with wave numbers from 500 to 4000 cm^–1. The FTY720-NOB-PLGA NPs underwent drug release at 1× PBST and 37 °C. Concentrations of FTY720 and NOB were determined at wavelengths of 200 and 330 nm and expressed as (B) grams of cumulative drug and (C) percent of total cumulative drug.

Figure 4

(A) Normal lung fibroblasts (IMR-90 cells) were treated with pure drug (NOB-(1) 2.5 × 10^–3 mg/mL and NOB-(2) 5 × 10^–3 mg/mL), drug combination (FTY20-NOB-(1) 4.06 × 10^–4 mg/mL FTY20 + 2.5 × 10^–3 mg/mL NOB and FTY20-NOB-(2) 8.125 × 10^–4 mg/mL FTY20 + 5 × 10^–3 mg/mL NOB) and PLGA encapsulated NPs (NOB-PLGA-NPs, FTY-NOB-PLGA). CCK-8 assay was used for measurement of viability. After stimulation with LPS for 24 h, Raw264.7 cells were treated with pure drug (NOB-(1) 5 × 10^–3 mg/mL and NOB-(2) 1 × 10^–2 mg/mL), drug combination (FTY20-NOB-(1) 8.125 × 10^–4 mg/mL FTY20 + 5 × 10^–3 mg/mL NOB and FTY20-NOB-(2) 1.625 × 10^–3 mg/mL FTY20 + 10^–2 mg/mL NOB) and PLGA encapsulated NPs (NOB-PLGA-NPs, FTY-NOB-PLGA NPs) to investigate their impact on secretion of IL-6 (B) and ROS (C) using ELISA assay and Ultra-Glo recombinant luciferase and d-cysteine assay, respectively. The values presented are the mean ± SD. p values were obtained using the Student’s t test (***p < 0.001, **p < 0.01 and *p < 0.05).

Figure 5

(A) Schematic diagram illustrating the design of the animal model. (B) Evaluation of IL-6 and TNF-α secretion in bronchoalveolar lavage fluid (BALF) from LPS-induced lung injury mice (n = 3 per group) after 24 h of treatment with PBS, NOB-PLGA NPs, and FTY720-NOB-PLGA NPs using an ELISA kit. (C) Histopathological examination of lung tissues in mice (n = 3 per group) with LPS-induced lung injury treated with PBS, NOB-PLGA NPs, and FTY720-NOB-PLGA NPs at 24 h, displayed at an original magnification of 100×. (D) Panoramic images of NF-κB obtained through the TissueFAXs platform and quantification of NF-κB expression percentage in the entire lung section (***p < 0.001, **p < 0.01, and *p < 0.05).

Figure 6

(A) Schematic diagram of the animal model examining the endurance of FTY720-NOB-PLGA NPs’ efficacy over a 48 h period. (B) Assessment of IL-6 and TNF-α secretion in bronchoalveolar lavage fluid (BALF) from LPS-induced lung injury mice (n = 3 per group) 48 h post-treatment with PBS, NOB-PLGA NPs, and FTY720-NOB-PLGA NPs using an ELISA kit. (C) Lung pathology evaluation in mice (n = 3 per group) with LPS-induced lung injury treated with different drugs at 48 h, displayed at an original magnification of 100×. (D) Panoramic images of NF-κB obtained through the TissueFAXs platform, along with quantification of NF-κB expression percentage in the entire lung section (***p < 0.001, **p < 0.01, and *p < 0.05).

Figure 7

(A) Schematic illustration of FTY720-NOB-PLGA NPs inhalation for 30 min daily over three consecutive days, followed by sacrifice at 96 h. (B) Comparison of the impact of different treatments (PBS and FTY720-NOB-PLGA NPs; 30 min/day for 3 days) on IL-6 and TNF-α secretion in BALF using an ELISA kit. (C) Lung pathology assessment in mice (n = 3 per group) with LPS-induced lung injury treated with different drugs for three consecutive days. (D) Panoramic NF-κB images obtained through the TissueFAXs platform, and quantification of NF-κB expression percentage in the entire lung section. Mean ± SD values are presented, and p-values were determined using the Student’s t test (***p < 0.001, **p < 0.01, and *p < 0.05).

Figure 8

Histological analysis of lung tissue sections from control, LPS-induced acute lung injury, and post-treatment with FTY720-NOB-PLGA NPs. (A) Tissue sections were stained using H&E and immunohistochemical staining for MPO, CD68, and CD3. Positive cells are brown (magnification, 400×). Scale bar, 10 μm. (B) Quantification of neutrophils (MPO), macrophages (CD68), and T cells (CD3) per high-power field (HPF) derived from IHC data (n = 3 per group). Data are presented as mean ± SD. Statistical significance was assessed using the Student’s t test (***p < 0.001, **p < 0.01).

Figure 9

Multiplexed mIF assay of immune cell infiltration. (A) Representative images from the mIF assay on lung tissue from mice post-LPS stimulation with or without FTY720-NOB-PLGA nanoparticle inhalation treatment, showing staining for MPO (neutrophils), CD68 (macrophages), CD3 (T cells), and DAPI (nuclei). Scale bar, 100 μm. (B) The bar graph illustrates the proportion of neutrophils, macrophages, and T cells infiltrating the lungs of mice following LPS stimulation with or without FTY720-NOB-PLGA nanoparticle inhalation treatment. Data are presented as mean ± SD, calculated from four fields per mouse lung. Statistical significance was determined using the Student’s t test (***p < 0.001).

Figure 10

Body weight, organ function, and respiratory rate in control group and treatment group. (A) Body weight percentage of mice in response to LPS and FTY720-NOB-PLGA NPs. The values presented are the mean ± SD. p values were obtained using the Student’s t test (***p < 0.001). (B) Blood biochemical results of the FTY720-NOB-PLGA NPs following inhalation into mice (n = 5). The results show mean and standard deviation of alanine aminotransferase (ALT), creatinine (CRE), total bilirubin (TBIL), and blood urea nitrogen (BUN). (C) Respiratory parameters: Mean of breathing frequency was analyzed during day 1 to day 3 through plethysmography associated with the Allay restrainer.