(W0930-06-36) Pulmonary Delivery of Drug-Loaded Superparamagnetic Iron Oxide Nanoparticles for Diagnosis and Treatment of Tuberculosis

Purpose: Tuberculosis (TB) remains a leading cause of global morbidity and mortality, further complicated by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Despite the availability of first-line therapies, conventional treatments are prolonged, systemically toxic, and often suffer from poor targeting efficiency, resulting in suboptimal patient compliance, increased toxicity, and high relapse rates. Moreover, diagnostic methods are limited by delayed detection, low sensitivity in paucibacillary cases, and inadequate accessibility in resource-limited settings, contributing to disease progression and ongoing transmission. Addressing these urgent clinical gaps necessitates the development of new drug delivery and diagnostic strategies. This research aimed to design a pulmonary theranostic platform composed of poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) coated superparamagnetic iron oxide nanoparticles (SPIONs) for the inhalational delivery of antitubercular agents. The platform was intended to achieve localized drug delivery to alveolar granulomas, minimize systemic side effects, enhance patient adherence, and enable real-time monitoring of therapeutic response via magnetic resonance imaging (MRI) contrast enhancement.

Methods: Superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized through a chemical co-precipitation method and surface-modified with oleic acid to ensure colloidal stability in organic and aqueous environments. These SPIONs were subsequently encapsulated within a biodegradable PLGA-PEG polymeric matrix using a double emulsion solvent evaporation technique, optimized for nanoparticle size and encapsulation efficiency. Two model therapeutic agents were incorporated: Ethambutol, a first-line antitubercular drug targeting arabinogalactan synthesis, and N-(adamantyl)-1H-indole-2-carboxamide (N2), a novel MmpL3 inhibitor disrupting mycolic acid transport in mycobacteria. Nanoparticles were characterized by dynamic light scattering (DLS) for hydrodynamic diameter and polydispersity, transmission electron microscopy (TEM) for morphology, powder X-ray diffraction (PXRD) for crystallinity confirmation, and thermogravimetric analysis (TGA) for quantifying iron content. LC-MS/MS was utilized to determine drug encapsulation efficiency and release kinetics. Magnetic properties, including saturation magnetization, coercivity, and blocking temperature, were evaluated using a magnetometer and T₁/T₂ relaxivity studies. The nanoparticles were freeze-dried with cryoprotectants to enable pulmonary delivery and analyzed using a Next Generation Impactor (NGI) to determine aerodynamic particle size distribution. In vitro cytotoxicity assays were performed on A549 lung carcinoma cells and BEAS-2B normal bronchial epithelial cells to evaluate biocompatibility. Extracellular antibacterial efficacy was assessed against Mycobacterium tuberculosis surrogate strains. Additionally, the uptake of these nanoparticles was studied using a THP-1 derived three-dimensional (3D) spheroid granuloma model to mimic the pathological microenvironment of pulmonary TB.

Results: The optimized PLGA-PEG coated SPION nanoparticles exhibited a hydrodynamic diameter of approximately 250 nm with a narrow polydispersity index, indicative of uniform particle size distribution and minimal aggregation. Zeta potential measurements revealed highly negative surface charges, confirming excellent colloidal stability. TEM imaging demonstrated uniform spherical morphology, while PXRD confirmed the magnetite crystalline phase without secondary impurities. Magnetic characterization revealed superparamagnetic behavior with negligible remanence and coercivity, and a blocking temperature near 50 K, indicating suitability for MRI applications. T₂ relaxivity measurements showed a high r₂ value of 73.709 s¹ · mM¹ and an r₂/r₁ ratio of 76.3, supporting their potential as a strong T₂-weighted MRI contrast agent. LC-MS/MS analysis showed 31.68% and 77.67% drug loading efficiencies for Ethambutol and N2, respectively, with sustained drug release profiles observed over 72 hours. Freeze-dried powders exhibited mass median aerodynamic diameters (MMADs) ranging from 2 and 4 μm, optimal for alveolar deposition in the deep lung. MTT assays indicated low cytotoxicity across all tested nanoparticles after 72 hours, demonstrating good biocompatibility. Antibacterial efficacy studies revealed inhibition of bacterial growth, validating the therapeutic potential of the drug-loaded nanoparticles. Moreover, a 3D THP-1 macrophage spheroid model was successfully developed.

Conclusion: This study successfully developed a multifunctional, inhalable nanoparticle system integrating therapeutic and diagnostic capabilities for pulmonary tuberculosis management. The PLGA-PEG encapsulated SPIONs demonstrated favorable physicochemical properties, including excellent colloidal stability, high drug loading efficiency, robust superparamagnetic behavior, and strong MRI contrast enhancement, achieving aerodynamic profiles suitable for deep lung targeting. The nanoparticles exhibited significant antibacterial efficacy and minimal cytotoxicity, underscoring their potential for safe and effective pulmonary delivery. Notably, the theranostic platform allows non-invasive monitoring of nanoparticle distribution and treatment response through MRI, addressing critical limitations in current TB therapy. A 3D M. tuberculosis granuloma model is being evaluated to examine further nanoparticle penetration, uptake, and therapeutic performance within structured granulomatous environments. Overall, this inhalable SPION-based theranostic system offers a promising strategy for improving tuberculosis treatment outcomes by enabling targeted delivery, reducing systemic toxicity, enhancing patient compliance, and facilitating real-time imaging of therapeutic efficacy.

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Acknowledgements: The authors thank Erik Moore (Lab Manager, Department of Pharmacy Sciences, School of Pharmacy and Health Professions), Dr. Justin Tolman (Associate Professor, Department of Pharmacy Sciences, School of Pharmacy and Health Professions), and Dr. Adrian Draney (Instrumentation Specialist, Adjunct Instructor, Department of Chemistry and Biochemistry, College of Arts and Sciences)

Funding: The authors thank the NIH R15 AI164443-01, 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.


Characterization of Nanoparticles


A) M/H Hysteresis Loop for OA-SPIONs, B) Magnetic Moment v/s Temperature (OA-SPIONs), C) T2 relaxation (60 MHz) of OA-SPIONs, and D) TEM images of OA-SPIONs


A) Cytotoxicity studies in BEAS-2B cells, B) Cytotoxicity studies of Ethambutol (ETB) and N2 in BEAS-2B cells, C) THP-1 Macrophages 3D Spheroids, and D) Cytotoxicity of OA-SPIONs in A549 cells.