(T1330-04-22) N-acetylcysteine-Loaded Magnetite Chitosan Microparticle (NMCP) Part Ⅱ: In Vitro and In Vivo Study to Confirm the Nitrate Adsorption Behavior

Purpose: In recent years, the problem of air pollution is emerging and the particulate matter (PM) is the main cause.   PMs are divided into two principal groups: coarse and fine particles with an aerodynamic diameter mostly larger and smaller than 2.5 µm, respectively. They are not discharge in naturally and it lead to lung diseases and long-term exposure of PMs could premature death. Nitrates in PM is important environmental toxicants and pose significant health risks. Therefore it is important to effectively adsorb nitrate introduced into the body and discharge it out of the lungs. For this reasons, N-acetylcysteine magnetite chitosan microparticle (NMCP) was manufactured as adsorption methods for the removal of anionic pollutants such as nitrate. The aim of this study was to confirmed the nitrate adsorption behavior of NMCP in-vitro and in-vivo.

Methods: In-vivo and in-vitro studies were evaluated by Fourier transform infrared spectroscopy (FT-IR), Raman microscope, Franz diffusion cell and pharmacokinetic study. Spectroscopic evaluation was subjected to attenuated total reflectance FT-IR spectroscopy in the range of 650-4000 cm-1 using an IFS 66/S spectrometer (BRUKER OPTIK GMBH, Germany). Raman imaging was done using a Raman microscope (RAMANtouch; Nanophoton) and the laser beam was focused onto the sample using a 100x objective (NA = 0.90, Nikon) with excitation at 532 nm. The intensity of the laser beam was ca. 5.3 x 104 W cm-2. Sample for raman imaging were prepared that particles were sprayed evenly using a dry powder insufflator (DP-4, Pennecnury TM), with a nitrate solution and then lyophilized. In-vitro nitrate permeation profile was evaluated using a Franz diffusion cell (FCDS-900C, Labfine Instruments, Anyang, Korea). The Franz diffusion cell was chosen to mimic the diffusion control air-liquid interface in the lungs. The medium was continuously stirred and cellulose membrane filter (Advantec, Tokyo, Japan) was used as a barrier. After permeation study of predetermined time point, obtained sample were analyzed by HPLC. Pharmacokinetics study for results of FT-IR and plasma concentration was conducted with 250-270 g of rats. The rats were anaesthetized and 500 μL blood was taken. Bronchoalveolar lavage fluid (BALF) and blood samples were analyzed by FT-IR and HPLC after the pretreatment process. All animal experiments were performed in accordance with the “Principles of Laboratory Animal Care” and were approved by the Committee for Animal Experiments of Chungbuk National University (Cheongju, Korea).

Results: In-vitro results of FT-IR, N-H bond was shifted from 1625 cm-1 to 1612 cm-1 which caused by the reaction of nitrate with chitosan amine group. Also the in-vivo results of BALF measured by FT-IR showed same as in-vitro. In the results of Raman microscope, nitrate attracted around NMCP and found that the intensity was high around NMCP. The attraction behaviors of NMCP could be visualized through raman image. In nitrate permeation test, there was no difference at permeation rate whether NMCP or not until 30 minutes. But after 30 minutes, permeation rate decreased rapidly when dose of NMCP higher. Final permeation rate is the smallest at high dose of NMCP. The results of pharmacokinetics study evaluating plasma nitrate concentration showed the higher amount of NMCP reduced AUC and Cmax more.

Conclusion: The nitrate adsorption behavior of NMCP was confirmed in in-vitro and in vivo studies. NMCP has been successfully designed to adsorb and remove nitrates in the lungs, especially from trachea and bronchi except alveoli, and can prevent the potential danger of nitrates contained in particulate matter from air pollution.

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).