## 3 Results and Discussion
As shown in Figure 6a,b, the micro-porous membrane filter was found to exhibit no cytotoxicity, with a strong green fluorescence signal, and no changes in the cell morphology, compared to the control for both the L-929 and A-549 cells. Furthermore, the cells under the micro-porous membrane filter retained their typical morphologies, as compared to the negative control (HDPE). However, the cellular morphologies of the positive control (ZEDC-PU) of both cells mostly changed significantly, with the green fluorescence signal being either weak or entirely absent. This indicated that the positive control of both cells completely died as a result of the ZDEC released from the positive control film. The extraction method also presented no significant cytotoxicity of the micro-porous membrane filter, with no decreased cell viabilities for both the L-929 and A-549 cells, as shown in Figure 6c,d. Figure 6: _Cont_. The results of the biocompatibility test suggested that the microporous membrane filter can be useful for the delivery of some drugs using a nebulizer. To generate the micro-aerosol drops, the Pd-Ni alloy membrane filter was mounted on the designed mock-up spray head, and a piezoelectric transducer was used to drive the ultrasonic nebulizer. The designed mock-up of the nebulizer device includes a medication chamber, piezoelectric actuator, and electrical control unit. Figure 7 shows the micro-nebulizer designed in this study. The volumetric flow rate of a droplet becomes the largest when the vibration frequency of the spray head approaches the frequency of mechanical resonance. The optimal vibration frequency of the spray head was the second resonance of 98.5 kHz (displacement: 0.0091), which is a high-frequency vibration operation. The result shows that when the working frequency of the piezoactuator is set at 98.5 kHz, a nebulizing flow rate of 0.34 mL/min is obtained. The electrical requirements of PZT were 14 V (rms) and 178 mA (rms). Therefore, power consumption was approximately 2.5 W.  
Figure 6: In vitro biocompatibility of the micro-porous membrane filter. (**a**) Images of green fluorescent protein (GFP)-transfected L-929 and (**b**) A-549 cells around and under the negative, positive, and filter samples. Magnified images in the insets were taken with a 4\(\times\) optical lens. Scale bars are 100 \(\upmu\)m. Quantitative analysis of cell viability of (**c**) L-929 and (**d**) A-549 cells in the extraction method. (\(n=4\), \({}^{*}p<0.05\) compared to control). Figure 7: Micro-porous mesh nebulizer developed in this study. Figure 8 shows the characteristic diagrams of aerosol drops of various drug solutions. The droplets of all drugs were mostly distributed between 1 \(\upmu\)m and 10 \(\upmu\)m (Figure 8). In the case of deionized (DI) water, the median value of the particle size distribution (Dv(50) value shown in Figure 8) showed that the average particle size of the nebulized drug was 4.53 \(\upmu\)m. In addition, the surface weighted mean (D[3][2] value shown in Figure 8), which is sensitive to a small particle size, was 1.09 \(\upmu\)m. This result indicated that it can be inferred that the droplet size distribution of the micro-nebulizer described in this research was finer and smaller than that of current and conventional devices. On the other hand, the Dv(50) value of amphetamine solution was 3.65 \(\upmu\)m and the D[3][2] value was 0.70 \(\upmu\)m. These results indicated that, in the case of the nebulized amphetamine solution, the average size of the particles, and that of small particles, was smaller than the particles of nebulized DI water. These differences may be caused by the formation of irregularly-aggregated amphetamine molecules at high concentrations of amphetamine. Generally, it is well known that organic drug molecules with amphiphilic properties participate in the formation of irregular aggregation at high concentrations. Therefore, the formed aggregates were expected to greatly affect the size shrinkage of drug droplets. Similar results were obtained for the levofloxacin solution.