(T1530-04-22) N-acetylcysteine-Loaded Magnetite Chitosan Microparticle (NMCP) Part I: Preparation of Inhalable Chitosan-Fe₃O₄ Composite for Removal of Nitrate of Particulate Matter

Purpose: In recent years, air pollution is threatening human health. It is estimated that more than 2.5 million deaths worldwide each year occur as a direct result of air pollution through the lung and respiratory damage. The main cause of air pollution is particulate matter (PM). Typically, nitrates in PM are mainly produced as secondary materials in the atmosphere following the oxidation of nitrogen oxides (NO and NO₂). Nitrate and nitrite compounds in PM are important environmental toxicants and pose significant health risks. The toxic effects of nitrate on the airways include bronchoconstriction, increased nonspecific airway responsiveness, and mucociliary dysfunction. The nitrates in the PM enter the body and are rapidly ionized and absorbed. PM is an invisible size of dust less than 2.5 μm that easily migrates to the deep lungs. It can cause diseases and cannot be removed from the lung. To discharge PM, we devised a microparticle using N-acetylcysteine (NAC). In this study, we prepared an inhalable NAC-loaded magnetite chitosan microparticle (NMCP) containing chitosan-Fe₃O₄ for adsorption of nitrate in particulate matter.

Methods: The particle preparation method in this study was based on one of the solvent diffusion emulsion methods. The formulation was designed by Design of Experiment (DoE) which employs the response surface curve of factors used in the Box-Behnken design (BBD). Scanning electron microscopy (SEM) and Focused ion beam (FIB) showed the morphology of chitosan-Fe₃O₄ composite microparticle. X-ray photoelectron spectroscopy (XPS) analyzed the atomic concentration of O, Fe, and S atoms along with estimated sputtering depth. Each atom measures Chitosan, Fe₃O₄, and NAC which consist of NMCP. The in vitro aerosol dispersion performance of NMCP was determined using an eight-stage non-viable Andersen Cascade Impactor (ACI) without a pre-separator (TE-20-800, TISCH Environmental, OH, USA) and with RS01^® (Plastiape, Germany) as dry powder inhaler device following USP Chapter < 601 > specification on aerosols.

Results: The BBD design was applied to investigate the effect of independent variables glutaraldehyde amount (X1), chitosan concentration (X₂), and the ratio of chitosan and Fe₃O₄ (X₃) on dependent variables loading content (Y₁) and nitrate adsorption amount (Y₂) for the optimization of NMCP. The predicted values of Y₁ and Y₂ were 11.2% and 23.5 μg/mg respectively, at X₁, X₂, and X₃ levels of 3 mL, 0.5%, and 1:0.1 (Figure 1). As a confirmation process, a fresh formulation of optimized NMCP was prepared with 0.5% Chitosan in the dispersion phase, the ratio of chitosan and Fe₃O₄ (1:0.1), and 3 mL glutaraldehyde. The optimized levels of factors yielded a formulation with a loading content of 13.7 ± 0.2% and nitrate adsorption amount of 18.9 ± 4.1 μg/mg. In the morphological observation of the NMCP, it was confirmed that the particles were generally round and occasionally had irregular shapes, and the surface of the particles was roughly formed. In the particles cut by FIB (Figure 2), it was confirmed that the particles were full from the surface to the inside, and as a unique point, shells of ~150 to ~300 nm were clearly distinguished from the surface of the particles. This was also the case in XPS results. When considering the composition of the element, it was confirmed that the shell from 0 to 300 nm is mainly composed of chitosan. Fe₃O₄ and NAC increase and chitosan decrease as it goes inside. The emitted dose for NMCP was 96.3 ± 0.6% which significantly high value. The fine particle fraction indicating the ability of particles to reach the respirable region with the median mass aerodynamic diameter (MMAD) of about 5.0 μm or less was 31.6 ± 0.8% for NMCP. The NMCP is about 5 μm, where the MMAD of the NMCP is the respirable aerodynamic particle size and exhibits excellent aerosol performance, optimally delivered to deep areas of the lung by sedimentation and diffusion (Figure 3)

Conclusion: NMCP formulations were prepared solvent diffusion method and optimized by DoE. In addition, the NMCP was designed with good inhalation efficiency so that particulate matter can be delivered to the deep lungs it reaches.

Acknowledgments: This study was supported by the National Research Foundation of Korea grants provided by the Korean government (NRF-2021R1A2C4002746 and 2017R1A5A2015541). This study was also supported by the Regional Innovation Strategy (RIS) through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (MOE).

Figure 1. Response surface plot according to formulation variance of Y1: loading content and Y2: nitrate adsorption amount



Figure 2. SEM images of the cut NMCP using a focused ion beam (A) Magnification ×3,000 (B) Magnification ×12,000. (C) X-ray photoelectron spectroscopy depth profile studies of NMCP.



Figure 3. Aerodynamic performance of NMCP by Andersen cascade impactor