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Inhalation February 2025 27 them ideal for delivering delicate drug formulations to the respiratory tract [36]. Advanced non-clinical testing approaches: Air-liquid interface (ALI) and precision cut lung slices (PCLS) e pharmaceutical industry recognizes transition- ing from non-clinical to clinical stages in drug devel- opment as challenging, therefore the development of robust cell culture models that avoid animal exper- iments is critical in drug development. Immortal- ized cell lines, such as the Caco-2 model, are crucial for pharmacokinetic studies and early-stage drug development, due to their ease of handling and cost- effectiveness [39]. For testing pulmonary deliv- ery, both immortalized and patient-derived primary cells can be used, with primary cultures offering disease-specific characteristics but being harder to reproduce. e 16HBE14o-, Calu-3, and A549 cell lines each have specific applications and limitations in mimicking lung tissue. e 16HBE14o- cell line, used in studying cystic fibrosis, forms polarized lay- ers but lacks cilia and tight junctions [40, 41]. e Calu-3 line, advantageous for mucus penetration studies, forms strong tight junctions under air- liquid interface (ALI) conditions, crucial for repli- cating the human respiratory tract [41]. e A549 line, resembling human AT2 cells, is used for distal ronment for drug absorption and delivery, due to their large surface area, thin epithelial barrier and extensive vascularization [28, 29]. However, several barriers must be overcome, such as mucus and surfactant, which hinder drug penetration. Mucus, a glycoprotein-rich gel, increases lung lining fluid viscosity, reducing drug diffusion [30-33]. Addi- tionally, pulmonary surfactant, composed mainly of phospholipids and proteins, forms a layer that can hinder drug stability and absorption. [34, 35]. For inhalation, various inhalable pharmaceutical devices are available, including pressurized metered dose inhalers, dry powder inhalers, soft mist inhalers, and nebulizers (jet, ultrasonic, and vibrating mesh) [28, 29]. Among these, vibrating mesh nebulizers (VMN) are particularly effective for shear-sensitive materials (such as polyplexes) due to their gentle delivery method, which maintains particle integrity and transfection efficiency. [36-38]. For local treatment of pulmonary diseases, including COVID-19, aerosolized nanoparticles are especially suitable. Inhalation devices such as VMNs ensure that the polymeric nanoparticles are exposed to mini- mal shear stress, maintaining their efficacy. Moreover, these devices facilitate targeted drug delivery to the lungs, optimizing therapeutic outcomes for respira- tory conditions. Studies have shown that VMNs cre- ate the mildest conditions for aerosolization, making Figure 3 Visualization of polyplex behavior in lung explants: Two photon acquisition of ex vivo poly(spermine acrylamides)/siRNA polyplex distribution in fibrotic human lung tissue (hPCLS) transfected with siGLO (in blue/cyan) at N/P 5 and marked with Hoechst (in red). Collagen I and II are indicated by the second harmonic generation (SHG) in orange. Panel A: transfection control with siGLO. Panel B: vehicle control without siGLO, reproduced from reference 8, which was distributed under the terms of the Creative Commons CC BY License. A B