Inhalable Formulation Based on Lipid-Polymer Hybrid Nanoparticles for the Macrophage Targeted Delivery of Roflumilast

Abstract

Here, novel lipid-polymer hybrid nanoparticles (LPHNPs), targeted to lung macrophages, were realized as potential carriers for Roflumilast administration in the management of chronic obstructive pulmonary disease (COPD). To achieve this, Roflumilast-loaded fluorescent polymeric nanoparticles, based on a polyaspartamide-polycaprolactone graft copolymer, and lipid vesicles, made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-(polyethylene glycol)-mannose, were properly combined using a two-step method, successfully obtaining Roflumilast-loaded hybrid fluorescent nanoparticles (Man-LPHFNPs@Roflumilast). These exhibit colloidal size and a negative ζ potential, 50 wt % phospholipids, and a core-shell-type morphology; they slowly release the entrapped drug in a simulated physiological fluid. The surface analysis also demonstrated their high surface PEG density, which confers mucus-penetrating properties. Man-LPHFNPs@Roflumilast show high cytocompatibility toward human bronchial epithelium cells and macrophages and are uptaken by the latter through an active mannose-mediated targeting process. To achieve an inhalable formulation, the nano-into-micro strategy was applied, encapsulating Man-LPHFNPs@Roflumilast in poly(vinyl alcohol)/leucine-based microparticles by spray-drying.

Scheme 1. Synthetic Route of the PHEA-g-RhB-g-Succ-PCL Graft Copolymer: PHEA (Black), RhB (Fuchsia), PCL (Red, n = 122), Succinic Residue (Succ, Green)

Reagents and conditions: (a) a-DMF, CDI, DEA, 5 h at 40 °C; (b) 68 h at 40 °C.

Figure 1

STEM image of Man-LPHFNPs. The bar represents 200 nm. One drop of the sample dispersion (3 mg/mL) was placed on a holey carbon-coated copper grid, air-dried overnight, and imaged using an SEM/STEM Fei-ThermoFisher Versa 3D.

Figure 2

XPS curve-fitting of the photoelectron peak N 1s of (A) FNPs and (B) Man-LPHFNPs. Spectra were recorded on each freeze-dried sample using a PHI 5000 VersaProbe II and monochromatic Al-Kα radiation (hv = 1486.6 eV) from an X-ray source operating at a spot size of 200 μm, a power of 50 W, and an acceleration voltage of 15 kV. The fitting was done using the model Gauss–Lorentz, Shirley background. The scattered yellow line refers to the raw data, while the solid red and blue lines refer to the curve-fitting results.

Figure 3

(A) Transmittance at 650 nm of dispersions containing mucin in the presence of Man-LPHFNPs [0.1 mg/mL blue; 0.5 mg/mL orange], LPHFNPs [0.1 mg/mL red; 0.5 mg/mL green], and chitosan [0.5 mg/mL, black]. (B) Complex viscosity of artificial mucus alone (black) and treated with LPHFNPs 0.1 mg/mL (red) and Man-LPHFNPs 0.1 mg/mL (blue).

Figure 4

Percentage of Roflumilast released by LPHFNPs (red), Man-LPHFNPs (blue), and diffusion profile (black). Roflumilast release profile from LPHFNPs was realized using the dialysis method, under sink conditions. Each sample was dispersed in 1 mL of PBS at pH 7.4, placed in a dialysis tubing (MWCO 12–14 kDa), immersed into PBS (pH 7.4)/ethanol (80/20, v/v), and then incubated at 37 °C under continuous stirring. At fixed time intervals (1, 2, 4, 6, and 24 h), an aliquot of the external medium was withdrawn, replaced with an equal volume of fresh medium, and freeze-dried, and Roflumilast was quantified by HPLC analysis as reported in the experimental part.

Figure 5

DSC thermogram of (A) FNPs, (B) DPPC/DSPE-PEG₂₀₀₀-mannose liposomes, (C) physical mixture of FNPs and liposomes, (D) Man-LPHFNPs, (E) Man-LPHFNPs@Roflumilast, and (F) Roflumilast in the crystalline form. Each sample (5–10 mg) was sealed in an aluminum pan and submitted to heating and cooling cycles in the temperature range of 20–300 °C at a scanning rate of 5 °C/min for heating and a scanning rate of 10 °C/min for cooling.

Figure 6

Cell viability assay after 24 h for (A) RAW 264.7 and (B) 16-HBE cells treated with free Roflumilast (black), LPHFNPs (pink), LPHFNPs@Roflumilast (red), Man-LPHFNPs (light blue), and Man-LPHFNPs@Roflumilast (blue). (*P < 0.05; **P < 0.001).

Figure 7

Fluorescence microscopy images of (A) RAW 264.7 cells and (B) 16-HBE cells treated after (a) 4 h and (b) 24 h of incubation. The bar represents 10 μm.

Figure 8

SEM images of NiM particles (sn 5000×, dx 66000×).