## 3 Results and Discussion
### Stability Study
#### 3.2.5 In Vitro Lung Deposition by Aerodynamic Measurements  
The deposition profile of the spray-dried samples was evaluated using Andersen Cascade Impactor (ACI), and the in vitro aerodynamic results were analyzed with Inhalytix(tm) V 2.0 software (Figure 9). The freshly prepared F1 formulation demonstrated a high fine particle fraction (FPF) (~64%), surpassing those of commercially available formulations used with the Breezhaler(r) device [30]. Additionally, the emitted fraction (EF) was notably high (~95%), reflecting efficient powder release from the inhaler. This enhanced aerosolization  
Figure 8: XRPD structural analysis of (**A**) raw ketoprofen (KTP) and the physical mixture (PM), and (**B**) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage. The circles highlight the characteristic peaks of KTP in the raw drug, physical mixture, as well as fresh and stored formulations.  
performance can be attributed to the inclusion of MNT and leucine, which improve powder cohesion and adhesion to hydroxypropyl methylcellulose (HPMC) capsules, ensuring consistent dose delivery [28].  
Although no significant changes in the MMAD were observed after storage, a decline in FPF values was recorded (Figure 9). This reduction can likely be attributed to surface morphology changes during storage, such as increased roughness or partial agglomeration due to moisture absorption under stressed conditions. The decrease in FPF suggests heightened cohesiveness among aerosol particles post-storage, potentially caused by moisture uptake, which increases their tendency to form agglomerates midair. Such changes may disrupt flow dynamics and reduce aerosolization efficiency. These findings align with those of a previous study by [PERSON] et al. (2020), which reported a significant decrease in FPF after storage while observing no substantial changes in MMAD [31].  
Figure 10 illustrates the particle distribution across the stages of the ACI. The ACI stages numbered \(-1\) to \(6\), are used to assess the particle size distribution of aerosolized formulations. Each stage corresponds to a specific cut-off diameter based on the aerodynamic diameter of the particles. The cut-off diameters (in \(\upmu\)m) for each stage are as follows: \(-1\): \(8.6\), \(-0\): \(6.5\), \(1\): \(4.4\), \(2\): \(3.2\), \(3\): \(1.9\), \(4\): \(1.2\), \(5\): \(0.55\), and \(6\): \(0.26\)[28]. Effective KTP deposition was predominantly observed in the second, third, and fourth stages, which correspond to the bronchial and upper alveolar regions [32]. A significant portion also reached the filter, representing deposition in the deeper lung regions, emphasizing the powder's capability to target smaller airways.  
An important aspect to consider is the impact of MNT concentration on aerodynamic characteristics. As demonstrated in our previous study [16], high MNT content can lead to crystallization, phase separation, and an increase in particle size, potentially compromising the aerosol performance of MNT-containing formulations. Consistent with these findings, [PERSON] et al. (2024) reported that MNT concentration significantly influenced the physical and aerosolization stability of spray-dried protein formulations [15]. However, assessing the specific effects of MNT concentration on the stability of the current formulation lies beyond the scope of this study.  
Figure 9: In vitro aerodynamic characteristics (MMAD, FPF, and EF) of sample F1 were analyzed as a freshly prepared sample (F1\(-\)0m), and after 1 (F1\(-\)1m) and 3 (F1\(-\)3m) months of storage, at a flow rate of 60 L/min. Data are presented as means \(\pm\) SD (\(n\) = 3 independent measurements). Statistical significance is indicated, ***** \(p\) \(<\) 0.0001 significantly different.  
To address the reduced aerodynamic performance after storage, future studies should explore different capsule materials and storage conditions, particularly varying relative humidity levels, to enhance stability and maintain aerosolization properties.