Spermidine-mediated poly(lactic- co-glycolic acid) nanoparticles containing fluorofenidone for the treatment of idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis is a progressive, fatal lung disease with poor survival. The advances made in deciphering this disease have led to the approval of different antifibrotic molecules, such as pirfenidone and nintedanib. An increasing number of studies with particles (liposomes, nanoparticles [NPs], microspheres, nanopolymersomes, and nanoliposomes) modified with different functional groups have demonstrated improvement in lung-targeted drug delivery. In the present study, we prepared, characterized, and evaluated spermidine (Spd)-modified poly(lactic-co-glycolic acid) (PLGA) NPs as carriers for fluorofenidone (AKF) to improve the antifibrotic efficacy of this drug in the lung. Spd-AKF-PLGA NPs were prepared and functionalized by modified solvent evaporation with Spd and polyethylene glycol (PEG)-PLGA groups. The size of Spd-AKF-PLGA NPs was 172.5±4.3 nm. AKF release from NPs was shown to fit the Higuchi model. A549 cellular uptake of an Spd-coumarin (Cou)-6-PLGA NP group was found to be almost twice as high as that of the Cou-6-PLGA NP group. Free Spd and difluoromethylornithine (DFMO) were preincubated in A549 cells to prove uptake of Spd-Cou-6-PLGA NPs via a polyamine-transport system. As a result, the uptake of Spd-Cou-6-PLGA NPs significantly decreased with increased Spd concentrations in incubation. At higher Spd concentrations of 50 and 500 µM, uptake of Spd-Cou-6-PLGA NPs reduced 0.34- and 0.49-fold from that without Spd pretreatment. After pretreatment with DFMO for 36 hours, cellular uptake of Spd-Cou-6-PLGA NPs reached 1.26-fold compared to the untreated DFMO group. In a biodistribution study, the drug-targeting index of Spd-AKF-PLGA NPs in the lung was 3.62- and 4.66-fold that of AKF-PLGA NPs and AKF solution, respectively. This suggested that Spd-AKF-PLGA NPs accumulated effectively in the lung. Lung-histopathology changes and collagen deposition were observed by H&E staining and Masson staining in an efficacy study. In the Spd-AKF-PLGA NP group, damage was further improved compared to the AKF-PLGA NP group and AKF-solution group. The results indicated that Spd-AKF-PLGA NPs are able to be effective nanocarriers for anti-pulmonary fibrosis therapy.

Keywords: fluorofenidone; idiopathic pulmonary fibrosis; nanoparticles; polyamine transport system; spermidine.

Figure 1

Construction of Spd-AKF-PLGA NPs. Abbreviations: Spd, spermidine; AKF, fluorofenidone; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS, N-hydroxysuccinimide.

Figure 2

¹H NMR spectra of PEG-PLGA copolymer in CDCl₃. Notes: (A) H₂N-PEG-COOH; (B) PLGA-COOH; (C) synthesized PEG-PLGA-COOH copolymer. Abbreviations: NMR, nuclear magnetic resonance; PEG, polyethylene glycol; PLGA, poly(lactic-co-glycolic acid).

Figure 2

¹H NMR spectra of PEG-PLGA copolymer in CDCl₃. Notes: (A) H₂N-PEG-COOH; (B) PLGA-COOH; (C) synthesized PEG-PLGA-COOH copolymer. Abbreviations: NMR, nuclear magnetic resonance; PEG, polyethylene glycol; PLGA, poly(lactic-co-glycolic acid).

Figure 3

Transmission electron microscopy of AKF-PLGA NPs (A) and Spd-AKF-PLGA NPs (B). Abbreviations: AKF, fluorofenidone; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; Spd, spermidine.

Figure 4

Release profiles of AKF solution, AKF-PLGA NPs, and Spd-AKF-PLGA NPs (n=3). Abbreviations: AKF, fluorofenidone; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; Spd, spermidine.

Figure 5

Fluorescence images of A549 cells following incubation with NPs at 0.5, 1 and 2 hours (n=6). Notes: After 0.5, 1 and 2 h incubation with a suspension of Cou6 (green)-containing PLGA NPs (200 µg/mL) at 37°, cellular action of A549 cells were stained with DAPI (blue) and examined using a fluorescent inverted microscope (magnification 40×). (A) Cou6-PLGA NPs at 0.5 h, (B) Spd-Cou6-PLGA NPs at 0.5 h, (C) Cou6-PLGA NPs at 1 h, (D) Spd-Cou6-PLGA NPs at 1 h, (E) Cou6-PLGA NPs at 2 h, and (F) Spd-Cou6-PLGA NPs at 2 hrs. Abbreviations: NPs, nanoparticles; Cou, coumarin; PLGA, poly(lactic-co-glycolic acid); Spd, spermidine; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride.

Figure 6

Fluorescence images of A549 cells following incubation with NPs (100, 200, and 400 µg/mL) at 1 hour (n=6). Notes: After 1 h incubation with a suspension of Cou6 (green)-containing PLGA NPs (100, 200, 400 µg/mL) at 37°, cellular action of A549 cells were staining with DAPI (blue) and examined using a fluorescent inverted microscope (magnification 40×). (A) 100 µg/mL Cou6-PLGA NPs, (B) 100 µg/mL Spd-Cou6-PLGA NPs, (C) 200 µg/mL Cou6-PLGA NPs, (D) 200 µg/mL Spd-Cou6-PLGA NPs, (E) 400 µg/mL Cou6-PLGA NPs, and (F) 400 µg/mL Spd-Cou6-PLGA NPs. Abbreviations: NPs, nanoparticles; Cou, coumarin; PLGA, poly(lactic-co-glycolic acid); Spd, spermidine; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride.

Figure 7

Flow-cytometry histograms of A549 cells following coincubation with NPs. Notes: (A) Blank control; (B) Cou6-PLGA NPs; (C) Spd-Cou6-PLGA NPs. Abbreviations: NPs, nanoparticles; Cou, coumarin; PLGA, poly(lactic-co-glycolic acid); Spd, spermidine.

Figure 8

Flow-cytometry histograms of A549 cells following the co-incubation with NPs and Spd. Notes: (A) Blank NPs; (B) Spd-Cou6-PLGA NPs; (C) 5 µM Spd + Spd-Cou6-PLGA NPs; (D) 50 µM Spd + Spd-Cou6-PLGA NPs; (E) 500 µM Spd + Spd-Cou6-PLGA NPs. Abbreviations: NPs, nanoparticles; Spd, spermidine; Cou, coumarin; PLGA, poly(lactic-co-glycolic acid); Geo mean, geometrical mean.

Figure 9

Effect of Spd on A549-cell uptake of Spd-Cou6-PLGA NPs (n=6). Notes: *P<0.05, 50 µM Spd + Spd-Cou6-PLGA NPs vs Spd-Cou6-PLGA NPs; **P<0.001, 500 µM Spd + Spd-Cou6-PLGA NPs vs Spd-Cou6-PLGA NPs. Abbreviations: Spd, spermidine; Cou, coumarin; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles.

Figure 10

Flow-cytometry histograms of A549 cells following coincubation with NPs and DFMO. Notes: (A) Blank NPs; (B) Cou6-PLGA NPs; (C) DFMO + Cou6-PLGA NPs; (D) Spd-Cou6-PLGA NPs; (E) DFMO + Spd-Cou6-PLGA NPs. Abbreviations: NPs, nanoparticles; DFMO, difluoromethylornithine; Cou, coumarin; PLGA, poly(lactic-co-glycolic acid); Spd, spermidine.

Figure 11

Effect of DFMO on A549-cell uptake of NPs (n=6). Notes: *P<0.05, Spd-Cou6-PLGA NPs vs Cou6-PLGA NPs; **P<0.05, DFMO + Spd-Cou6-PLGA NPs vs Spd-Cou6-PLGA NPs. Abbreviations: DFMO, difluoromethylornithine; NPs, nanoparticles; Spd, spermidine; Cou, coumarin; PLGA, poly(lactic-co-glycolic acid).

Figure 12

In vivo fluorescence imaging of mouse lung after injection of Spd-DiR-PLGA NPs (A) and DiR-PLGA NPs (B) into the tail vein at 1, 3, 6, 24, and 48 hours (n=6). Abbreviations: Spd, spermidine; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles.

Figure 13

Lung fluorescence intensity–time curves for mice after injection of DiR-PLGA NPs and Spd-DiR-PLGA NPs into the tail vein (n=6). Abbreviations: PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; Spd, spermidine.

Figure 14

Concentration of AKF in blood, heart, liver, spleen, lung, and kidney. Notes: At 1, 3, 6, 12, 24, and 48 hours after intravenous administration of (A) AKF solution (30 mg/kg), (B) AKF NPs, and (C) Spd-AKF NPs in rats. (D) Concentration of AKF in lung of rats with AKF solution, AKF NPs, and Spd-AKF NPs, at different time points (mean ± SD, n=5). Abbreviations: AKF, fluorofenidone; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; Spd, spermidine.

Figure 15

H&E stains (A) and Masson staining (B) of lung sections at days 7, 14, 21, and 28 following different treatments. Notes: (A) NS; (B) PQ; (C) PD solution; (D) AKF solution; (E) AKF NPs; (F) Spd-AKF-PLGA NPs. Abbreviations: NS, normal saline; PQ, paraquat; PD, pirfenidone; AKF, fluorofenidone; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; Spd, spermidine.