A Novel Combined Dry Powder Inhaler Comprising Nanosized Ketoprofen-Embedded Mannitol-Coated Microparticles for Pulmonary Inflammations: Development, In Vitro-In Silico Characterization, and Cell Line Evaluation

Abstract

Pulmonary inflammations such as chronic obstructive pulmonary disease and cystic fibrosis are widespread and can be fatal, especially when they are characterized by abnormal mucus accumulation. Inhaled corticosteroids are commonly used for lung inflammations despite their considerable side effects. By utilizing particle engineering techniques, a combined dry powder inhaler (DPI) comprising nanosized ketoprofen-embedded mannitol-coated microparticles was developed. A nanoembedded microparticle system means a novel advance in pulmonary delivery by enhancing local pulmonary deposition while avoiding clearance mechanisms. Ketoprofen, a poorly water-soluble anti-inflammatory drug, was dispersed in the stabilizer solution and then homogenized by ultraturrax. Following this, a ketoprofen-containing nanosuspension was produced by wet-media milling. Furthermore, co-spray drying was conducted with L-leucine (dispersity enhancer) and mannitol (coating and mucuactive agent). Particle size, morphology, dissolution, permeation, viscosity, in vitro and in silico deposition, cytotoxicity, and anti-inflammatory effect were investigated. The particle size of the ketoprofen-containing nanosuspension was ~230 nm. SEM images of the spray-dried powder displayed wrinkled, coated, and nearly spherical particles with a final size of ~2 µm (nano-in-micro), which is optimal for pulmonary delivery. The mannitol-containing samples decreased the viscosity of 10% mucin solution. The results of the mass median aerodynamic diameter (2.4-4.5 µm), fine particle fraction (56-71%), permeation (five-fold enhancement), and dissolution (80% release in 5 min) confirmed that the system is ideal for local inhalation. All samples showed a significant anti-inflammatory effect and decreased IL-6 on the LPS-treated U937 cell line with low cytotoxicity. Hence, developing an innovative combined DPI comprising ketoprofen and mannitol by employing a nano-in-micro approach is a potential treatment for lung inflammations.

Keywords: combination product; inflammation; ketoprofen; mannitol; milling; nano-in-micro; particle engineering; pulmonary delivery; spray-drying.

Figure 1

Holding time as short-term stability (four weeks) of ketoprofen-containing nanosuspension (KN), characterized by PS, PDI, and ZP at two temperatures: (A) +4 °C and (B) 25 °C.

Figure 2

Morphology images using scanning electron microscope (SEM): (A) spray-dried samples and the diameter of final product measured by Image-J software; (B) spray-dried blanks (PVA_SDS, PVA_SDS_LEU, PVA_SDS_MAN, and PVA_SDS_LEU_MAN). PS: particle size. Data are mean ± SD (n = 3 independent measurements).

Figure 2

Morphology images using scanning electron microscope (SEM): (A) spray-dried samples and the diameter of final product measured by Image-J software; (B) spray-dried blanks (PVA_SDS, PVA_SDS_LEU, PVA_SDS_MAN, and PVA_SDS_LEU_MAN). PS: particle size. Data are mean ± SD (n = 3 independent measurements).

Figure 3

Contact angle, surface energy, polarity, and cohesion work. CAw: contact angle in water, CAd: contact angle in diiodomethane, Υ: surface energy, Υp: polar part, Υ: dispersive part, Wc: cohesion work. Results are expressed as mean ± SD (n = 3 independent measurements). Level of significance compared to KETO (* p < 0.05), (** p < 0.01).

Figure 4

Thermal and structural analysis of raw materials, spray-dried samples, and physical mixture. (A) DSC, (B) water content by TGA, and (C) XRPD. PM: physical mixture.

Figure 4

Thermal and structural analysis of raw materials, spray-dried samples, and physical mixture. (A) DSC, (B) water content by TGA, and (C) XRPD. PM: physical mixture.

Figure 5

Aerosol performance characterization: (A) In vitro distribution of spray-dried samples by Andersen Cascade Impactor (ACI); (B) in silico results of deposited mass of spray-dried samples. BH: breath holding time, ET: extra-thoracic, EXH: exhaled. Results are expressed as mean ± SD (n = 3 independent measurements).

Figure 6

In vitro release profile of raw KETO and spray-dried samples in simulated lung media, pH = 7.4. Results are expressed as mean ± SD (n = 3 independent measurements).

Figure 7

MTT viability study. Percentage of cell viability at different concentrations on (A) U937 and (B) A549 cells. Results are expressed as mean ± SD (n = 4 independent measurements).

Figure 8

Relative expression of IL-6 on two cell lines: (A) U937 and (B) A549. Control is the untreated cell line, LPS is the treated cell line, KETO, F0, F0.5, F1, and F2 are treated with LPS. Results are expressed as mean ± SD (n = 3 independent measurements). Level of significance: (* p < 0.05), (** p < 0.01).

Figure 9

Preparation methods using particle engineering techniques: (A) preparation of the pre-dispersion (ketoprofen-containing nanosuspension), (B) preparation of nanosized ketoprofen-embedded mannitol-coated microparticles as combined dry powder for inhalation. KETO: ketoprofen, MAN: mannitol, LEU: leucine.