(T1530-09-55) A Therapeutic Strategy with Inhalable Polydopamine-Based Nanocarrier for Nintedanib in Managing Idiopathic Pulmonary Fibrosis

Purpose: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with a median survival time of 3-5 years.^[1] According to the literature, the first-line antifibrotic drug, nintedanib (NIN), could effectively suppress the upregulation of inflammatory factors in epithelial cells and reactive oxygen species (ROS) generation to slow down disease progression. However, the low bioavailability and high incidence of gastrointestinal adverse effects of the oral nintedanib medication (Ofev^®) greatly restrain its clinical application. In this regard, developing inhaled nintedanib therapy may hold greater promise in realizing the true therapeutic potential of NIN in managing IPF. In recent years, mesoporous polydopamine (MPDA) nanoparticles, exhibiting excellent biosafety profile and drug loading capacity, are emerging nanodrug carriers.^[2] In this work, a design of experiments (DOE) approach with Box Box-Behnken Design (BBD) was first utilized to optimize the preparation of NIN encapsulated MDPA nanoparticles (NIN@MPDA). Then, the ROS scavenging ability of the optimized formulation with the highest NIN loading capacity and smallest particle size was evaluated using bleomycin-injured bronchiolar epithelial cells. Superior ROS scavenging effect was noted with NIN@MPDA compared with free NIN solution. Further development of NIN@MPDA for pulmonary delivery is warranted to improve the efficacy of NIN.

Methods: MPDA nanoparticles were first prepared via emulsion-induced interfacial polymerization (Figure 1a). F-127 polymer (0.3 mg/mL) and 3,3',5,5'-Tetramethylbenzidine were dissolved in a water: methanol (1:1, v:v) mixed solvent as a template. Then the tris buffer was added to create a basic environment. After 15-min stirring, dopamine solution was added dropwise to a final concentration of 0.01 mg/mL. After overnight reaction, blank MDPA was dispersed by probe sonication. The particle size was characterized by DLS and TEM. NIN was loaded via probe sonication. Key process parameters, including the methanol ratio of the NIN solution, drug concentration, and sonication time—were optimized by BBD(software: Design-Expert, version 13.0). Free NIN was removed by ultracentrifugation (14,000 × g, 90 min), and the unencapsulated drug quantified by HPLC. The encapsulation efficiency (EE) of NIN was calculated as follows: EE% = (total drug − unencapsulated drug)/total drug ×100%. A cellular pulmonary fibrosis model was established by co-incubating BEAS-2B cells with bleomycin (BLM) for 12 h. Cell viability was measured by CCK-8 assay. Cellular senescence was assessed with a senescence-associated β-galactosidase (SA-β-gal) staining kit, while intracellular ROS levels in BLM-induced cells after treatments were measured by DCFH-DA assay kit. Stained cells were observed by fluorescence microscopy.

Results: Characterization of blank MDPA nanoparticles:MPDA were successfully prepared with an average size of 263.2±2.55 nm (Figure 1b), which remained stable at least for 5 days under refrigeration (Figure 1c). TEM images further revealed that the MPDA nanoparticles were homogeneous spheres (Figure 1d). The prepared MDPA exhibited an excellent biological safety profile with no significant cytotoxicity to the BEAS-2B cell at a concentration of 100 µg/ml (Figure 1e). Optimization of NIN-loading through DOE: Methanol ratio of NIN solution, sonication time and drug ratio identified to play critical roles in controlling the particle size and EE in preliminary experiments were optimized with BBD (Figure 2a-c). Within the pre-set ranges, the size significantly increased at the two extreme conditions (highest drug mass with shortest sonication time and lowest drug mass with longest sonication time). As for the EE, lower methanol proportion in the NIN solution generally yielded higher values. The optimal conditions in preparing NIN@MDPA were predicted to be drug mass of 290.3 μg, sonication time of 3 min and methanol proportion of 55% to give a size of 186.35 nm and EE of 76.22%. NIN@MDPA was then synthesized under the suggested conditions. The experimental size and EE were 253.2 ± 6.55 nm and 66.8% ± 8.2%, comparable with the predicted values. ROS scavenging effect of nanoparticles in vitro: BLM induced damage to BEAS-2B epithelial cells in a dose-dependent manner (Figure 3a) and obvious aging tendency (Figure 3b) at a BLM concentration of 100 μM. Using this cellular model, we investigated the ROS scavenging ability of the optimized NIN@MPDA. From the fluorescence microscopy results (Figure 3c), the epithelial cells showed obvious green signal after BLM induction, and the intensity of green signal was significantly decreased after 12 h incubation with free NIN and MPDA/NIN@MPDA, indicating the ROS consumption of the treatment. Comparing the two treatment groups, NIN@MPDA had a weaker green signal but further quantifiable measurements are required to confirm the observation.

Conclusion: In this work, a dopamine-based drug delivery system with good drug-loading efficiency that can reverse the fibrosis of lung epithelial cells was developed. Through DOE, the nanoparticles could obtain suitable particle size and encapsulation efficiency. The nanoparticles exhibited an effective anti-fibrotic effect at cellular level. Besides, our preliminary results indicate that MPDA may have the effect of assisting to improve the anti-inflammatory effect of NIN, serving as a promising carrier for NIN in treating IPF.

References: [1] Hongwei, X., Ying, Z., Haotian, Z., Yunran, Z., Qingqing, X., Junya, L., Shuaipeng, F., Xinyi, L., Siling, W., & Qinfu, Z. (2023). Smart polydopamine-based nanoplatforms for biomedical applications: state-of-art and further perspectives. Coordination Chemistry Reviews, 488, 215153.
[2] Hutchinson, J., Fogarty, A., Hubbard, R., & McKeever, T. (2015). Global incidence and mortality of idiopathic pulmonary fibrosis: a systematic review. European Respiratory Journal, 46(3), 795-806.

Acknowledgements: The authors gratefully acknowledge the provision of CUHK graduate studentship to Yifan LUO

The profiles of dopamine-based nanoparticles. a) Illustration of preparation of nanoparticles by emulsion-induced interface polymerization. b) The TEM images of the nanoparticles. Scale bar = 50 nm. c) The size of the nanoparticles. d) The change of the nanoparticle size within 5 days e) The cell- viability of BEAS-2b cells after co-incubating with nanoparticles.

The profiles of optimization of drug-loading procedure through DOE a) The table of experimental ranges and levels of independent factors selected for particle size and encapsulation efficiency of nanoparticle preparation. b) Response surface plot showing the effects of Drug weight and sonication time on particle size. c) Response surface plot showing the effects of MeOH proportion and Drug weight on Encapsulation efficiency.

ROS scavenging effects of nanoparticles in vitro a) Cell viability of BEAS-2b cells treated with different concentrations of BLM, (n = 3). b) SA-β-gal detection in BEAS-2b cells with or without BLM induction, the black arrow indicating the blue staining of senescence-associated β-galactosidase, scale bars = 100 µm. c) ROS staining of BEAS-2b cells with BLM induction and different treatment, scale bars= 200 μm.