Search Results (20)

Background/Objectives: Transforming Growth Factor-beta (TGFβ1) plays a core role in the process of pulmonary fibrosis (PF). The progression of pulmonary fibrosis can be alleviated by siRNA-based inhibiting TGF-β1. However, the limitations of naked siRNA lead to the failure of achieving therapeutic effect. This study aimed to design lipid nanoparticles (LNPs) that can deliver siTGF-β1 to the lungs for therapeutic purposes. Methods: The cytotoxicity and transfection assay in vitro were used to screen ionizable lipids (ILs). Design of Experiments (DOE) was used to obtain novel LNPs that can enhance resistance to atomization shear forces. Meanwhile, the impact of LNPs encapsulating siTGF-β1 (siTGFβ1-LNPs) on PF was investigated. Results: When DLin-DMA-MC3 (MC3) was used as the ILs, the lipid phase ratio was MC3:DSPC:DMG-PEG2000:cholesterol = 50:10:3:37, and N/P = 3.25; the siTGFβ1-LNPs could be stably delivered to the lungs via converting the siTGFβ1-LNPs solution into an aerosol (atomization). In vitro experiments have confirmed that siTGFβ1-LNPs have high safety, high encapsulation, and can promote cellular uptake and endosomal escape. In addition, siTGFβ1-LNPs significantly reduced inflammatory infiltration and attenuated deposition of extracellular matrix (ECM) and protected the lung tissue from the toxicity of bleomycin (BLM) without causing systemic toxicity. Conclusions: The siTGFβ1-LNPs can be effectively delivered to the lungs, resulting in the silencing of TGF-β1 mRNA and the inhibition of the epithelial–mesenchymal transition pathway, thereby delaying the process of PF, which provides a new method for the treatment and intervention of PF. Full article

There is increased interest in developing non-animal test systems for inhalation exposure safety assessments. However, defined methodologies are absent for predicting local respiratory effects from inhalation exposure to irritants. The current study introduces a concept for applying in vitro and in silico methods for inhalation exposure safety assessment. Three in vitro systems, representing the upper (MucilAir™—nasal epithelial tissue) and lower (A549 cells and human precision-cut lung slices) human respiratory regions, were exposed to six respiratory irritants. These irritant exposures were conducted as liquid droplets, aerosol, or vapors, and samples were collected over 24 h. Cytotoxicity, cytokine release, epithelial resistance, oxidative stress, and mitochondrial membrane potential were measured. To determine the human relevance of in vitro exposures, airway surface depositions were predicted by simulating airborne concentrations equivalent to the Cramer class III inhalation threshold of toxicological concern limit of 0.47 mg/person/day using an in silico model. A > 100-fold margin of exposure was calculated comparing lowest concentrations showing in vitro effects to in silico simulated values. While further studies are needed, this manuscript presents a basic requirement for employing non-animal methods to inform inhalation exposure safety assessments by combining in vitro and in silico assays. Full article

Tuberculosis (TB) is an airborne bacterial infection caused by Mycobacterium tuberculosis (M. tb), resulting in approximately 1.3 million deaths in 2022 worldwide. Oral therapy with anti-TB drugs often fails to achieve therapeutic concentrations at the primary infection site (lungs). In this study, we developed a dry powder inhalable formulation (DPI) of clofazimine (CFZ) to provide localized drug delivery and minimize systemic adverse effects. Poly (lactic acid-co-glycolic acid) (PLGA) microparticles (MPs) containing CFZ were developed through a single emulsion solvent evaporation technique. Clofazimine microparticles (CFZ MPs) displayed entrapment efficiency and drug loading of 66.40 ± 2.22 %w/w and 33.06 ± 1.45 µg/mg, respectively. To facilitate pulmonary administration, MPs suspension was spray-dried to yield a dry powder formulation (CFZ SD MPs). Spray drying had no influence on particle size (~1 µm), zeta potential (−31.42 mV), and entrapment efficiency. Solid state analysis (PXRD and DSC) of CFZ SD MPs studies demonstrated encapsulation of the drug in the polymer. The drug release studies showed a sustained drug release. The optimized formulation exhibited excellent aerosolization properties, suggesting effective deposition in the deeper lung region. The in vitro antibacterial studies against H37Ra revealed improved (eight-fold) efficacy of spray-dried formulation in comparison to free drug. Hence, clofazimine dry powder formulation presents immense potential for the treatment of tuberculosis with localized pulmonary delivery and improved patient compliance. Full article

Non-small cell lung cancer (NSCLC) is a global concern as one of the leading causes of cancer deaths. The treatment options for NSCLC are limited to systemic chemotherapy, administered either orally or intravenously, with no local chemotherapies to target NSCLC. In this study, we have prepared nanoemulsions of tyrosine kinase inhibitor (TKI), erlotinib, using the single step, continuous manufacturing, and easily scalable hot melt extrusion (HME) technique without additional size reduction step. The formulated nanoemulsions were optimized and evaluated for their physiochemical properties, in vitro aerosol deposition behavior, and therapeutic activity against NSCLC cell lines both in vitro and ex vivo. The optimized nanoemulsion showed suitable aerosolization characteristics for deep lung deposition. The in vitro anti-cancer activity was tested against the NSCLC A549 cell line which exhibited 2.8-fold lower IC₅₀ for erlotinib-loaded nanoemulsion, as compared to erlotinib-free solution. Furthermore, ex vivo studies using a 3D spheroid model also revealed higher efficacy of erlotinib-loaded nanoemulsion against NSCLC. Hence, inhalable nanoemulsion can be considered as a potential therapeutic approach for the local lung delivery of erlotinib to NSCLC. Full article

The effectiveness of inhalation therapy depends on aerosol size distribution, which determines the penetration and regional deposition of drug in the lungs. As the size of droplets inhaled from medical nebulizers varies depending on the physicochemical properties of the nebulized liquid, it can be adjusted by adding some compounds as viscosity modifiers (VMs) of a liquid drug. Natural polysaccharides have been recently proposed for this purpose and while they are biocompatible and generally recognized as safe (GRAS), their direct influence of the pulmonary structures is unknown. This work studied the direct influence of three natural VMs (sodium hyaluronate, xanthan gum, and agar) on the surface activity of the pulmonary surfactant (PS) measured in vitro using the oscillating drop method. The results allowed for comparing the variations of the dynamic surface tension during breathing-like oscillations of the gas/liquid interface with the PS, and the viscoelastic response of this system, as reflected by the hysteresis of the surface tension. The analysis was done using quantitative parameters, i.e., stability index (SI), normalized hysteresis area (HAn), and loss angle (φ), depending on the oscillation frequency (f). It was also found that, typically, SI is in the range of 0.15–0.3 and increases nonlinearly with f, while φ slightly decreases. The effect of NaCl ions on the interfacial properties of PS was noted, which was usually positive for the size of hysteresis with an HAn value up to 2.5 mN/m. All VMs in general were shown to have only a minor effect on the dynamic interfacial properties of PS, suggesting the potential safety of the tested compounds as functional additives in medical nebulization. The results also demonstrated relationships between the parameters typically used in the analysis of PS dynamics (i.e., HAn and SI) and dilatational rheological properties of the interface, allowing for easier interpretation of such data. Full article

Anthropogenic activities and industrialization render continuous human exposure to semi-volatile organic compounds (SVOCs) inevitable. Occupational monitoring and safety implementations consider the inhalation exposure of SVOCs as critically relevant. Due to the inherent properties of SVOCs as gas/particle mixtures, risk assessment strategies should consider particle size-segregated SVOC association and the relevance of released gas phase fractions. We constructed an in vitro air–liquid interface (ALI) exposure system to study the distinct toxic effects of the gas and particle phases of the model SVOC dibutyl phthalate (DBP) in A549 human lung epithelial cells. Cytotoxicity was evaluated and genotoxic effects were measured by the alkaline and enzyme versions of the comet assay. Deposited doses were assessed by model calculations and chemical analysis using liquid chromatography tandem mass spectrometry. The novel ALI exposure system was successfully implemented and revealed the distinct genotoxic effects of the gas and particle phases of DBP. The empirical measurements of cellular deposition and the model calculations of the DBP particle phase were concordant.The model SVOC DBP showed that inferred oxidative DNA damage may be attributed to particle-related effects. While pure gas phase exposure may follow a distinct mechanism of genotoxicity, the contribution of the gas phase to total aerosol was comparably low. Full article

The clinical outcomes of lung cancer remain poor, mainly due to the chemoresistance and low bioavailability of systemically delivered drugs. Therefore, novel therapeutic strategies are urgently needed. The TNF-related apoptosis-inducing ligand (TRAIL)-armed extracellular vesicle (EV-T) has proven to be highly synergistic for the killing of cancer cells with the potent cyclin-dependent kinase (CDK) inhibitor Dinaciclib (Dina). However, both optimal drug formulations and delivery strategies are yet to be established to facilitate the clinical application of the combination of EV-T and Dina. We hypothesize that Dina can be encapsulated into EV-T to produce a complexed formulation, designated EV-T-Dina, which can be nebulized for pulmonary delivery to treat lung cancer with potentially improved efficacy and safety. The prepared EV-T-Dina shows good stability both in vitro and in vivo and is very efficient at killing two highly TRAIL-resistant cancer lines. The ability to overcome TRAIL resistance is associated with the concomitant downregulation of the expression of cFLIP, MCL-1, and Survivin by Dina. The EV-T-Dina solution is nebulized for inhalation, showing unique deposition in animal lungs and importantly it demonstrates a significant suppression of the growth of orthotopic A549 tumors without any detectable adverse side events. In conclusion, the aerosolized EV-T-Dina constitutes a novel therapy, which is highly effective and safe for the treatment of lung cancers. Full article

Effective pulmonary drug delivery using a metered-dose inhaler (MDI) requires a match between the MDI sprays, the patient’s breathing, and respiratory physiology. Different inhalers generate aerosols with distinct aerosol sizes and speeds, which require specific breathing coordination to achieve optimized delivery efficiency. Inability to perform the instructed breathing maneuver is one of the frequently reported issues during MDI applications; however, their effects on MDI dosimetry are unclear. The objective of this study is to systemically evaluate the effects of breathing depths on regional deposition in the respiratory tract using a ProAir-HFA inhaler. An integrated inhaler mouth-throat-lung geometry model was developed that extends to the ninth bifurcation (G9). Large-eddy simulation (LES) was used to compute the airflow dynamics due to concurrent inhalation and orifice flows. The discrete-phase Lagrangian model was used to track droplet motions. Experimental measurements of ProAir spray droplet sizes and speeds were used as initial and boundary conditions to develop the computational model for ProAir-pulmonary drug delivery. The time-varying spray plume from a ProAir-HFA inhaler into the open air was visualized using a high-speed imaging system and was further used to validate the computational model. The inhalation dosimetry of ProAir spray droplets in the respiratory tract was compared among five breathing depths on a regional, sub-regional, and local basis. The results show remarkable differences in airflow dynamics within the MDI mouthpiece and the droplet deposition distribution in the oral cavity. The inhalation depth had a positive relationship with the deposition in the mouth and a negative relationship with the deposition in the five lobes beyond G9 (small airways). The highest delivery efficiency to small airways was highest at 15 L/min and declined with an increasing inhalation depth. The drug loss inside the MDI was maximal at 45–60 L/min. Comparisons to previous experimental and numerical studies revealed a high dosimetry sensitivity to the inhaler type and patient breathing condition. Considering the appropriate inhalation waveform, spray actuation time, and spray properties (size and velocity) is essential to accurately predict inhalation dosimetry from MDIs. The results highlight the importance of personalized inhalation therapy to match the patient’s breathing patterns for optimal delivery efficiencies. Further complimentary in vitro or in vivo experiments are needed to validate the enhanced pulmonary delivery at 15 L/min. Full article

Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl⁻ currents and intracellular Ca²⁺ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca²⁺ activated Cl⁻ channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca²⁺ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application. Full article

Extensive production and use of nanomaterials (NMs), such as titanium dioxide (TiO₂), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO₂ NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO₂ NMs. Moreover, agglomerates are of prime importance in occupational exposure scenarios, but their toxicological relevance remains poorly understood. Therefore, the aim of this study was to investigate the potential pulmonary effects induced by TiO₂ agglomerates of different sizes at the air–liquid interface (ALI), which is more realistic in terms of inhalation exposure, and compare it to results previously obtained under submerged conditions. A nano-TiO₂ (17 nm) and a non-nano TiO₂ (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO₂ particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO₂ agglomerates at the ALI. At the end of 4h exposure, cytotoxicity, glutathione depletion, and DNA damage were evaluated. Our results indicate that dose deposition and the toxic potential in HBE cells are influenced by agglomeration and exposure via the ALI induces different cellular responses than in submerged systems. We conclude that the agglomeration state is crucial in the assessment of pulmonary effects of NMs. Full article

Inhalation therapy offers several advantages in respiratory disease treatment. Azithromycin is a macrolide antibiotic with poor solubility and bioavailability but with a high potential to be used to fight lung infections. The main objective of this study was to generate a new inhalable dry powder azithromycin formulation. To this end, an electrospray was used, yielding a particle size around 2.5 µm, which is considered suitable to achieve total deposition in the respiratory system. The physicochemical properties and morphology of the obtained microparticles were analysed with a battery of characterization techniques. In vitro deposition assays were evaluated after aerosolization of the powder at constant flow rate (100 L/min) and the consideration of the simulation of two different realistic breathing profiles (healthy and chronic obstructive pulmonary disease (COPD) patients) into a next generation impactor (NGI). The formulation was effective in vitro against two types of bacteria, Staphylococcus aureus and Pseudomonas aeruginosa. Finally, the particles were biocompatible, as evidenced by tests on the alveolar cell line (A549) and bronchial cell line (Calu-3). Full article

Excessive architectural re-modeling of tissues in pulmonary fibrosis due to proliferation of myofibroblasts and deposition of extracellular matrix adversely affects the elasticity of the alveoli and lung function. Progressively destructive chronic inflammatory disease, therefore, necessitates safe and effective non-invasive airway delivery that can reach deep alveoli, restore the surfactant function and reduce oxidative stress. We designed an endogenous surfactant-based liposomal delivery system of naringin to be delivered as an aerosol that supports pulmonary mechanics for the management of pulmonary fibrosis. Phosphatidylcholine-based liposomes showed 91.5 ± 2.4% encapsulation of naringin, with a mean size of 171.4 ± 5.8 nm and zeta potential of −15.5 ± 1.3 mV. Liposomes with the unilamellar structure were found to be spherical and homogeneous in shape using electron microscope imaging. The formulation showed surface tension of 32.6 ± 0.96 mN/m and was able to maintain airway patency of 97 ± 2.5% for a 120 s test period ensuring the effective opening of lung capillaries and deep lung delivery. In vitro lung deposition utilizing Twin Stage Impinger showed 79 ± 1.5% deposition in lower airways, and Anderson Cascade Impactor deposition revealed a mass median aerodynamic diameter of 2.35 ± 1.02 μm for the aerosolized formulation. In vivo efficacy of the developed formulation was analyzed in bleomycin-induced lung fibrosis model in rats after administration by the inhalation route. Lactate dehydrogenase activity, total protein content, and inflammatory cell infiltration in broncho-alveolar lavage fluid were substantially reduced by liposomal naringin. Oxidative stress was minimized as observed from levels of antioxidant enzymes. Masson’s Trichrome staining of lung tissue revealed significant amelioration of histological changes and lesser deposition of collagen. Overall results indicated the therapeutic potential of the developed non-invasive aerosol formulation for the effective management of pulmonary fibrosis. Full article

The nasal cavity is an attractive route for both local and systemic drug delivery and holds great potential for access to the brain via the olfactory region, an area where the blood–brain barrier (BBB) is effectively absent. However, the olfactory region is located at the roof of the nasal cavity and only represents ~5–7% of the epithelial surface area, presenting significant challenges for the deposition of drug molecules for nose to brain drug delivery (NTBDD). Aerosolized particles have the potential to be directed to the olfactory region, but their specific deposition within this area is confounded by a complex combination of factors, which include the properties of the formulation, the delivery device and how it is used, and differences in inter-patient physiology. In this review, an in-depth examination of these different factors is provided in relation to both in vitro and in vivo studies and how advances in the fabrication of nasal cast models and analysis of aerosol deposition can be utilized to predict in vivo outcomes more accurately. The challenges faced in assessing the nasal deposition of aerosolized particles within the paediatric population are specifically considered, representing an unmet need for nasal and NTBDD to treat CNS disorders. Full article

Invasive Pulmonary Aspergillosis (IPA) and Pneumocystis jiroveci Pneumonia (PCP) are serious fungal pulmonary diseases for immunocompromised patients. The brand name drug CANCIDAS^® (Caspofungin acetate for injection) is FDA approved to treat IPA, but is only 40% effective. Efficacious drug levels at the lung infection site are not achieved by systemic administration. Increasing the dose leads to toxicity. The objective, here, is to reformulate caspofungin for aerosolization to high drug concentration by lung targeted delivery and avoid systemic distribution. Described in this paper is a new, room temperature-stable formulation that meets these goals. The in vitro antifungal activity, solid state and reconstituted stability, and aerosol properties of the new formulation are presented. In addition, pharmacokinetic parameters and tissue distribution data are determined from nose-only inhalation studies in rats. Plasma and tissue samples were analyzed by High Performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS-MS). Inhaled drug concentrations for caspofungin Active Pharmaceutical Ingredient (API), and the new formulation, were compared at the same dose. In the lungs, the parameters C_max and Area Under Curve (AUC) showed a 70%, and 60%, respective increase in drug deposition for the new formulation without significant systemic distribution. Moreover, the calculated pharmacodynamic indices suggest an improvement in efficacy. These findings warrant further animal toxicology studies and human clinical trials, with inhaled caspofungin, for treating IPA. Full article

Pulmonary drug delivery represents an attractive, non-invasive administration option. In addition to locally acting drugs, molecules that are intended to produce systemic effects can be delivered via the pulmonary route. Several factors need to be considered in the context of delivering drugs to or via the lungs—in addition to the drug itself, its formulation into an appropriate inhalable dosage form of sufficient stability is critical. It is also essential that this formulation is paired with a suitable inhaler device, which generates an aerosol of a particle/droplet size that ensures deposition in the desired region of the respiratory tract. Lastly, the patient’s (patho-) physiology and inhalation manoeuvre are of importance. This Special Issue brings together recent advances in the areas of inhalation device testing, aerosol formulation development, use of in vitro and in silico models in pulmonary drug deposition and drug disposition studies, and pulmonary delivery of complex drugs, such as vaccines, antibiotics and peptides, to or via the lungs. Full article