2-Oxoglutarate-dependent non-heme iron oxygenases (2OGX) catalyze a broad spectrum of oxidative transformations, including hydroxylation, halogenation, desaturation, cyclization, rearrangement, and endoperoxidation, through a conserved HxD/E…H facial triad and Fe(IV)=O chemistry. Their functional diversity arises from structural elements that define the catalytic pocket. Substrate-binding architectures can be categorized into four recurrent motifs—the conserved lip (CLip), conserved lid (CLid), specific lid (SL), and dimer lid (DL)—together with the major β-sheet framework (βI–βVI); mutations in these lid/lip elements and within β-strands collectively govern substrate entry, positioning, and radical partitioning. This review discusses representative case studies organized by reaction class—hydroxylation/halogenation, cyclization/rearrangement, endoperoxidation, and free amino acid oxidation—to illustrate how targeted substitutions in these motifs enable rational reprogramming of reactivity. Examples include hydroxylases converted to halogenases, fungal enzymes redirected to construct alternative meroterpenoid scaffolds, endoperoxidases generating non-natural products, and amino acid hydroxylases engineered for halogenation, desaturation, or aziridination. These studies highlight the structural plasticity of 2OGX scaffolds and establish them as programmable biocatalysts, with advances in structural biology and computational design expected to accelerate their application in synthetic biology, natural product discovery, and drug development. The literature published from 2015 through September 2025 is reviewed.

Biologics represent a major advance in therapy, offering highly specific and potent treatment options for diseases previously difficult to manage; however, their administration routes are commonly limited to injection due to poor oral bioavailability, which can lead to patient inconvenience and adherence challenges. The pulmonary route offers a promising alternative, leveraging the lung’s large absorptive surface area, thin alveolar-capillary barrier, rich vascularization, and avoidance of first-pass metabolism to enable both local and systemic delivery. Effective inhaled biologic therapies require harmonizing the drug’s physicochemical properties with aerosol aerodynamic behavior and dissolution. The pharmacokinetic fate of inhaled biologics is further influenced by lung physiology, including airflow dynamics, airway structure, mucociliary clearance, and respiratory lining fluid composition. These factors present significant barriers to the stability, absorption, and retention of inhaled biologics. Extensive research efforts focus on optimizing formulations, inhalation devices, and excipients, alongside deepening the understanding of the biopharmaceutical characteristics of inhaled biologics. This review summarizes recent advances in inhalation systems of therapeutic peptides and proteins for systemic and local effects, emphasizing practical strategies to overcome key biopharmaceutical and physicochemical challenges, thus advancing the clinical potential of next-generation inhaled biologics.

Inhalation delivery of protein therapeutics has emerged as a promising non-invasive alternative to traditional injectable formulations that offers potential for both localized and systemic treatment of pulmonary diseases. This review comprehensively summarizes the current advances in inhalable protein formulations, with emphasis on design strategies, formulation technologies, barriers to effective delivery, and disease-specific applications. Key aspects include the role of particle size, surface charge, and protein engineering in optimizing lung deposition and cellular uptake, as well as techniques such as spray freeze drying and PEGylation to enhance protein stability. The review also explores novel therapeutic approaches that target cystic fibrosis, asthma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung infections, and cancer, including the use of antibodies, nanobodies, exosomes, and albumin-based carriers. Clinical translation remains limited, but ongoing innovation in delivery systems and molecular design is thought to hold significant promise for expanding the therapeutic landscape of inhaled protein drugs.

This study aimed to develop inhalable dry powder formulations of naked plasmid DNA (pDNA) for pulmonary gene delivery using an electrospinning (ES) technique. Nanofiber mats comprising polyvinyl alcohol (PVA), pDNA encoding firefly luciferase, and either D(−)-mannitol (Man) or lactose monohydrate (Lac) were fabricated and subsequently cryomilled into fine, respirable particles. Agarose gel electrophoresis revealed partial degradation of pDNA during both ES and milling processes, with Lac-based nanofiber mat and powder showing greater pDNA integrity than Man-based formulations. Intratracheal administration of the ES-derived powders in mice led to successful in vivo gene expression, with Man-based powders milled for 0.5 min yielding the highest luciferase activity. Pulmonary imaging using indocyanine green showed that dry powders exhibited extended lung residence compared to aqueous formulations, likely due to improved mucosal adhesion and slower dissolution. Remarkably, the ES-generated pDNA powders demonstrated superior transfection efficiency over both naked pDNA and pDNA–polyethyleneimine complexes, despite some loss in pDNA integrity. These findings highlight the importance of dispersibility and lung retention in achieving effective pulmonary gene transfer. The ES approach represents a promising platform for producing inhalable pDNA powders, offering a non-invasive gene therapy option for respiratory diseases.

The structural integrity of inhalable liposomal carriers is a key determinant of drug release behavior, pulmonary residence time, and therapeutic efficacy. This study aimed to establish a compensated Förster resonance energy transfer (FRET)-based platform for the quantitative assessment of liposome integrity throughout the inhalation delivery process. By compensation for donor fluorescence bleed-through and direct acceptor excitation, the method enables accurate quantification of FRET signals from liposomes co-loaded with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD, donor)/1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR, acceptor), using both spectrofluorometry and macroscopic imaging. Upon treatment with Triton X-100, decreases in the compensated FRET/donor ratio were detected at lower concentrations than those required to induce measurable changes in membrane anisotropy and within the same concentration range as the onset of encapsulated 6-carboxyfluorescein release. Using this platform, in vitro aerosol characterization with an Andersen cascade impactor enabled simultaneous analysis of aerodynamic particle size distribution and stage-specific liposome integrity. In vivo experiments in mice—including ex vivo lung imaging and bronchoalveolar lavage fluid analysis—revealed a time-dependent decline in liposome integrity during pulmonary residence. This ability to monitor carrier structural stability from initial deposition through residence—an aspect not readily achieved with conventional single-fluorophore labeling—offers significant advantages for formulation development, stabilization strategies, and dosing regimen optimization. The FRET-based platform could, in principle, be adapted for use with other standardized cascade impactors such as the Next Generation Impactor and is expected to be applicable to a wide range of lipid-based or polymeric nanocarriers, including inhalable vaccines and nucleic acid therapeutics.

Mucociliary clearance is a unique defense mechanism that forcibly transfers micron-sized substances deposited on the airway epithelium from the lungs, but may interfere with inhalation therapy. The application of mucoadhesive agents to inhaled formulations is expected to improve their efficacy by prolonging the drug residence time in the lungs through inhibited mucociliary clearance. In the present study, we attempted to develop new inhaled spray-freeze-dried (SFD) powders with high deagglomeration and pulmonary retention abilities by combining hydroxypropyl cellulose (HPC) of different molecular weights as a mucoadhesive agent and dileucine (diLeu) as a dispersion enhancer. The incorporation of diLeu into SFD powders resulted in a rough surface structure with large cavities and improved their deagglomeration abilities. In addition, the incorporation of HPC of lower molecular weights resulted in SFD powders with higher deagglomeration abilities. On the other hand, SFD powders with HPC and diLeu showed similar particle size distributions to that with trehalose (Tre) and diLeu after emission from a device regardless of the molecular weight of HPC incorporated. A biodistribution study through intratracheal administration to mice revealed that pulmonary drug retention was longer with SFD powders with HPC and diLeu than with a drug solution or SFD powder with Tre and diLeu, and also that HPC of a high molecular weight (approx.100000) provided the highest pulmonary retention ability of the SFD powder. These results strongly indicate that the molecular weight of HPC incorporated is a critical factor that markedly affects both the deagglomeration and pulmonary retention abilities of SFD powders.