(M1430-09-51) Aerosolizable Drug Loaded Superparamagnetic Iron Oxide Nanoparticle Theranostic for Pulmonary Tuberculosis

Purpose: Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) are resistant to first-line drugs, including isoniazid and rifampicin, and present significant diagnostic and treatment challenges. There is an emerging need for improved diagnostic tools and therapies for this debilitating disease (1). This can be achieved by a theranostic approach, which simultaneously provides real-time diagnostics and treatment options. Superparamagnetic iron oxide nanoparticles (SPIONs) are synthetic maghemite or magnetite particles with sizes between 1 and 100 nm, which can improve diagnostics by enhancing magnetic resonance imaging (MRI) for real-time monitoring during early detection and can be used for targeted delivery via Inhalation (2). Such a multifunctional approach offers several advantages, such as direct lung targeting, higher local drug concentration, and reduced systemic side effects, making it more effective and patient-friendly (3). However, this approach has some limitations, including the limited drug-loading capability of SPIONs and distribution for effective lung penetration. These challenges can be met by using additional polymeric coating and further functionalization using mucus-penetrating PLGA-PEG co-polymer for improved drug loading and targeted delivery (4,5). This study aims to develop an inhalation delivery system using drug-loaded SPIONs to improve the targeted delivery of antitubercular drugs N2 (N-(adamantyl)-1H-indole-2-carboxamide) and ethambutol in the diagnosis and treatment of pulmonary TB.

Methods: SPIONs were synthesized via alkaline coprecipitation method by mixing Fe⁺³ and Fe⁺² salts in a molar ratio of 2:1, respectively, with 2.5 M NaOH. This reaction resulted in black magnetite (Fe3O4) SPIONs under an inert nitrogen atmosphere, which was stabilized using oleic acid. SPIONs were further coated with PLGA using a double emulsion method to enhance drug loading capability. Briefly, stabilized SPIONS and PLGA were dissolved in dichloromethane, probe sonicated for one minute, followed by addition of 2% PVA solution. This mixture was probe sonicated for two minutes, resulting in an emulsion that was washed with deionized water to remove impurities. The cytotoxic effects of oleic acid coated SPIONs were tested on the A549 lung carcinoma cell line using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ethambutol was obtained commercially, but N2 was synthesized in our lab. N2 was synthesized by coupling indole-2-carboxylic acid and 1-adamantylamine using HOBt (1-hydroxybenzotriazole) and 3-eimethylamino-propyl)-ethyl-carbodiimide hydrochloride (EDC). Product formation was monitored using thin-layer chromatography, and the final product was isolated using flash chromatography and characterized using ¹H NMR spectroscopy and mass spectrometry. A liquid chromatography-tandem mass spectrometry (LCMSMS) method was developed to quantify drug loading and in vitro release. Separation was performed using a 5 µm C-18 100 Å column (50 × 3.0 mm). A step gradient of 10% acetonitrile and 90% water was used to elute ethambutol for the first 100 seconds, followed by 70% acetonitrile and 30% water from 100 to 180 seconds to elute N2. The flow rate was set at 0.5 mL/min. The SPION/ethambutol-loaded PLGA-PEG nanoparticles were prepared using the emulsion solvent evaporation technique. The primary organic phase (O1) consisted of oleic acid stabilized SPIONs dispersed in chloroform, and the water phase (W1) consisted of ethambutol dissolved in DI water that formed the inner aqueous phase. These two immiscible phases were mixed in a ratio of 5:2, stirred at room temperature for one hour, and probe sonicated for 30 seconds under an ice bath. The secondary oil phase (O2) consisted of PLGA-PEG in chloroform and was combined with the (O1/W1) emulsion and probe sonicated for 30 seconds. Finally, this emulsion was mixed with 3% PVA aqueous phase (W2) and probe sonicated to form the multiple emulsion (O1/W1/O2/W2). These formulations were characterized using dynamic light scattering (DLS) for particle size, polydispersity, and zeta potential. Their morphology and size were further assessed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

Results: The particle size, polydispersity, and zeta potential for uncoated, oleic acid-stabilized, PLGA-coated, and Ethambutol-loaded PLGA-PEG SPIONs are listed in Table 1. SEM images (Figure 1A) confirm the spherical shape of PLGA-coated nanoparticles. TEM images (Figures 1B, 1C, and 1D) show the actual diameter of oleic acid-stabilized SPIONs as approximately 11.45 nm and confirm the formation of ethambutol-loaded PLGA-PEG SPIONs. The LC method was able to separate both drugs with a retention time of 0.38 minutes for ethambutol and 2.73 minutes for N2. The standard curves for ethambutol and N2 were linear over the concentration ranges of 1.95-125 ng/mL and 0.97-125 ng/mL, respectively. Figures 2A and 2B illustrate cell viability at various concentrations of oleic acid coated SPIONs.

Conclusion: The study shows successful formulation and characterization of SPIONs in nano-size ranges. Based on TEM analysis as the particle diameter is less than 25 nm, these magnetic nanoparticles can be considered superparamagnetic. The cytotoxicity assay showed that these SPIONs have no significant cytotoxic effect over the concentration range of 0.25 mg/mL to 5 mg/mL for 48 and 72 hours, respectively.

References: 1. Dheda K, Mirzayev F, Cirillo DM, Udwadia Z, Dooley KE, Chang KC, et al. Multidrug-resistant tuberculosis. Nat Rev Dis Primer. 2024 Mar 24;10(1):1–27.
2. Zarepour A, Zarrabi A, Khosravi A. SPIONs as Nano-Theranostics Agents. In: Zarepour A, Zarrabi A, Khosravi A, editors. SPIONs as Nano-Theranostics Agents [Internet]. Singapore: Springer; 2017 [cited 2024 Jul 16]. p. 1–44. Available from: https://doi.org/10.1007/978-981-10-3563-0_1
3. McBride AA, Price DN, Lamoureux LR, Elmaoued AA, Vargas JM, Adolphi NL, et al. Preparation and Characterization of Novel Magnetic Nano-in-Microparticles for Site-Specific Pulmonary Drug Delivery. Mol Pharm. 2013 Oct 7;10(10):3574–81.
4. Schneider CS, Xu Q, Boylan NJ, Chisholm J, Tang BC, Schuster BS, et al. Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation. Sci Adv. 2017 Apr 5;3(4):e1601556.
5. Luque-Michel E, Lemaire L, Blanco-Prieto MJ. SPION and doxorubicin-loaded polymeric nanocarriers for glioblastoma theranostics. Drug Deliv Transl Res. 2021 Apr 1;11(2):515–23.

Acknowledgements: Acknowledgments: The authors thank Erik Moore, Lab Manager, Department of Pharmacy Sciences, School of Pharmacy and Health Professions.

Funding: The authors thank the Department of Pharmacy Sciences, Creighton University, for the financial support.
Conflict of interest statement: We confirm that there are no conflicts of interest associated with this work, and there has been no significant financial support for this work that could have influenced its outcome.

Table 1: Characterization of SPION formulations

Figure 1: A) SEM image of PLGA coated SPIONs, B) TEM image of Oleic acid stabilized SPION, C&D) TEM image of Ethambutol/PLGA-PEG SPIONs


Figure 2: A) Percent cell viability versus concentration of SPIONs at 48 hours, B) Percent cell viability versus concentration of SPIONs at 72 hours.