Design, development, and preclinical evaluation of pirfenidone-loaded nanostructured lipid carriers for pulmonary delivery

Abstract

Pirfenidone is an antifibrotic and anti-inflammatory drug used for the management of idiopathic pulmonary fibrosis. The current oral delivery of PD has multiple drawbacks, including first-pass metabolism and gastrointestinal discomfort. Efforts have been made to create nanostructured lipid carriers (NLCs) using solid lipids, liquid lipids, and surfactants through an emulsification process followed by ultrasonication to achieve sustained drug release. A central composite design (CCD) utilizing response surface methods (RSMs) was employed to develop and optimize the formulation. The assessed characteristics included particle size distribution, surface topography, drug entrapment efficiency, in vitro drug release, and kinetic profiles in animal models. Cytotoxicity experiments were performed on HepG2 and Caco-2 cell lines and compared with that of PD-NLCs. The optimized formulation yielded a particle size of 159.8 ± 3.46 nm and an encapsulation efficiency of 81.4 ± 7.1% after 10 freeze-thaw cycles of homogenized lipid carriers. In vitro tests assessing various tested flow rates revealed that over 95% of the released drug was retrieved. In vitro studies showed that the PD-loaded nanostructured lipid carrier (NLC) was more cytotoxic to HepG2 and Caco-2 cells than a pure aqueous solution of the drug. Using 25% w/w sorbitol as a cryoprotectant, the findings showed no variation in the properties of NLC before and after freeze-drying. PD-NLCs carriers were shown to have better bioavailability, longer retention time in the lung, and a 15.94-targeting factor related to the PD aqueous solution. Hence, the outcomes confirmed the potential of the PD-NLCs formulation to improve the efficacy of the drug in inhalation therapy.

Keywords: Bioavailability; Caco2; Central composite design; HepG2; Lung targeting; Nanostructured lipid carriers; Pirfenidone; Pulmokinetic parameters.

Fig. 1

Overview of present study.

Fig. 2

Solubility determination of PD in various solid lipids, liquid lipids and surfactants.

Fig. 3

counter and 3D-response surface plot showing the effect of independent variables on particle size.

Fig. 4

counter and 3D response surface plots showing the effect of independent variables on Entrapment Efficiency,

Fig. 5

The actual and predicted value of (A) particle size as Y1, (B) encapsulation efficiency as Y2, and (C) Overly plot of dependent variables.

Fig. 6

(A) Particle size and (B) zeta potential of the improved PD-NLCs (F5).

Fig. 7

Morphological characterization of Optimized NLCs (F5) (A) SEM, (B) TEM, and (C) AFM.

Fig. 8

(A) FTIR spectroscopy of (1) Drug, (2) Stearic acid, (3) Oleic acid, (4) Tween 20, (5) Physical Mixture, and (6) Formulation. (B) DSC thermographs of (1) Drug, (2) Physical Mixture, and (3) Optimized NLCs formulation (F5). (C) Powdered X-Ray diffractogram of 1. Drug, 5. Physical Mixture, and 7. Optimized NLCs formulation (F5)

Fig. 9

PD release from PD-aqueous solution and PD-NLCs in vitro. The data averages three repetitions, with the error bar indicating the standard deviation.

Fig. 10

Cytotoxicity on Caco-2 cell line (% vs. untreated cells) of (a) NLCs and (b) PD-NLCs at different dilutions (1:100, 1:150, 1:200, and 1:300) after 3 h of exposure. Data are shown as mean ± standard error (n = 3). * = p < 0.05.

Fig. 11

In vitro viability of PD-NLCs and PD in A549 cells. The MTT assay examines the cytotoxicity of PD-aqueous solution and PD-NLCs at various cell doses.

Fig. 12

(A) Aqueous PD solution and PD-NLCs were intratracheally injected into Wistar rats for 24 h, and PD concentrations in lung homogenate were determined (LH). (B) PD concentration in BAL. Mean Standard Deviation (SD) for n = 6 data points.