# Freeze-Drying Ethlycellulose Microparticles Loaded with
## 3 Results and Discussion  
### Preparation and Characterization of the Prepared Ethlycellulose Microparticles Loaded with ETO  
The main objective of this study was to prepare a microparticle formulation with improved solubility and enhanced release in media at pH 7.4 (suitable duodenal bulb medium). Microparticles composed of EC containing ETO with drug-polymer ratios of 1:1, 1:3, 1:6, and 1:20 were prepared with regular sizes and spherical shapes, and the sizes obtained by LPSA are shown in Table 1. Table 1 shows that the microparticles loaded with ETO and dried by lyophilization had a smaller size than the same formulation dried by oven-drying. The blank lyophilized microparticles had a nano range of 86.1 \(\pm\) 0.01 nm in comparison to the dried microparticles' range of 2300 \(\pm\) 1012 nm. Figure 2a,b shows the primary dried microparticles prepared at a 1:1 of D/P containing ETO, which had a size of 163 um. The freeze-dried microparticles loaded with ETO showed a size of 0.088 um; the size was reduced due to lyophilization. [PERSON] et al. showed these same results, with the particle size of BSA decreasing as the PEG-BSA ratio increased due to freezing-induced condensation [26]. Moreover, it was previously reported that freeze-drying can produce smaller particle sizes with relatively large geometric diameters [3].  
\begin{table}
\begin{tabular}{c c c} \hline
**Drug to Polymer Ratio** & **Size of Dried (Lyophilized)** & **Size of Dried (Primary)** \\
**(ETO:EC)** & **ET-ETO MPs (μm)** & **ET-ETO MPs (μm)** \\ \hline blank & 0.086.1 \(\pm\) 0.01 & 2.3 \(\pm\) 1.12 \\
1:1 & 0.088 \(\pm\) 0.001 & 163.1 \(\pm\) 2.3 \\
1:3 & 1.77 \(\pm\) 0.012 & 189.6 \(\pm\) 0.2 \\
1:6 & 6.33 \(\pm\) 0.08 & 222.1 \(\pm\) 12 \\
1:20 & 91.5 \(\pm\) 0.11 & 234 \(\pm\) 2.3 \\ \hline \end{tabular}
\end{table}
Table 1: The different sizes of dried ethylcellulose microparticles loaded with etoposide that were prepared and dried by primary or freeze-drying.  
The spherical shape of the prepared microparticles was observed in the photos obtained from scanning electron microscopy (SEM) (Figure 3). The pictures revealed that both the primary dried and freeze-dried samples were spherical (Figure 3a-c). Particularly, the freeze-dried samples vary in morphology and size (Figure 3d) from the primary-dried samples, as they are porous and rough with sticky shill-like shapes (Figure 3e,f). The microparticles maintained their form after freeze-drying, which indicates that freeze-dried microparticles were not affected by the process of lyophilization and were still spherical (Figure 3d-f) [27,28].  
Figure 2: Average size distribution of the prepared ethylcellulose microparticles, blank microparticles (EC-MPs), and loaded etoposide microparticles (ET–ETO-MPs) determined using a particle size analyser: (**a**) mean particle size distribution and (**b**) three-dimensional graph of the EC microparticles.  
The SEM results agreed with the results obtained from LPSA and confirmed that the size of the ET-ETO microparticles was higher than that of the empty microparticles, as shown in Figure 3. The observed difference in size was due to the difference in shape obtained by SEM. It has also been reported that particle size may increase after freeze-drying due to their aggregation [28; 29]. This also explains the difference in size observed in the SEM images and by LPSA, which confirmed the presence of irregular particle sizes after lyophilization. This finding is in agreement with a previous study which showed that the aggregated particles lead to an increased mean diameter after freeze-drying due to the shrinkage observed for the ET-ETO microparticles [30].