Loading of Beclomethasone in Liposomes and Hyalurosomes Improved with Mucin as Effective Approach to Counteract the Oxidative Stress Generated by Cigarette Smoke Extract

Abstract

In this work beclomethasone dipropionate was loaded into liposomes and hyalurosomes modified with mucin to improve the ability of the payload to counteract the oxidative stress and involved damages caused by cigarette smoke in the airway. The vesicles were prepared by dispersing all components in the appropriate vehicle and sonicating them, thus avoiding the use of organic solvents. Unilamellar and bilamellar vesicles small in size (~117 nm), homogeneously dispersed (polydispersity index lower than 0.22) and negatively charged (~-11 mV), were obtained. Moreover, these vesicle dispersions were stable for five months at room temperature (~25 °C). In vitro studies performed using the Next Generation Impactor confirmed the suitability of the formulations to be nebulized as they were capable of reaching the last stages of the impactor that mimic the deeper airways, thus improving the deposition of beclomethasone in the target site. Further, biocompatibility studies performed by using 16HBE bronchial epithelial cells confirmed the high biocompatibility and safety of all the vesicles. Among the tested formulations, only mucin-hyalurosomes were capable of effectively counteracting the production of reactive oxygen species (ROS) induced by cigarette smoke extract, suggesting that this formulation may represent a promising tool to reduce the damaging effects of cigarette smoke in the lung tissues, thus reducing the pathogenesis of cigarette smoke-associated diseases such as chronic obstructive pulmonary disease, emphysema, and cancer.

Keywords: 16HBE cells; beclomethasone; cigarette smoke extract; mucin; oxidative stress; phospholipid vesicles; pulmonary delivery.

Figure 1

Representative Cryo-TEM images of beclomethasone dipropionate loaded liposomes (A), mucin-liposomes (B), hyalurosomes (C) and mucin-hyalurosomes (D).

Figure 2

Average diameter and polydispersity index of vesicles stored for 5 months at room temperature (~25 °C). Mean values ± standard deviations are reported (n = 3).

Figure 3

Cell viability of 16HBE incubated for 24 h with beclomethasone dipropionate in dispersion (E) or loaded in liposomes (A), Mucin-liposomes (B), Hyalurosomes (C) and Mucin-hyalurosomes (D). Mean values ± standard deviations (error bars) are reported (n = 6).

Figure 4

ROS expression in unstressed cells or cells stressed with cigarette smoke extract (20%) and treated with beclomethasone dipropionate in dispersion or loaded in vesicles (10⁻⁹ M). Mean values ± standard deviations (error bars) are reported (n = 3). Symbol (*) indicates values statistically different (p < 0.05) to that of stressed and untreated cells.