# Freeze-Drying Ethlycellulose Microparticles Loaded with
## 3 Results and Discussion
### Cytotoxicity Assay  
An in vitro cytotoxicity assay of free ETO and the freeze-dried microparticles with a drug-polymer ratio of 1:1 was selected for evaluation using a method described previously with some modifications [41]. This test was performed in two mammalian cancer cell types, MCF-7 (bone cancer of the mammary organ) and Caco-2 (mammalian epithelial colorectal adenocarcinoma) (Figure 8). The IC\({}_{50}\) values for the free ETO and freeze-dried ET-ETO microparticles were 45.57 and 33.32 \(\upmu\)M in MCF-7 cells, respectively, while these values were 27.1 and 18.5 \(\upmu\)M for free ETO and freeze-dried ET-ETO microparticles in Caco-2 cells, respectively. Indeed, the results showed less cytotoxic activity for the freeze-dried ET-ETO microparticles compared to free ETO against both tested cell lines. These results suggest that the ETO coating with EC enhanced the activity of ETO by forming a barrier layer. These findings indicate that EC microparticles loaded with ETO can target a specific area in the gastrointestinal tract at pH 7.4 with potential anticancer activity. Importantly, the activities of the ET-ETO microparticles were synergistic with those of the free drugs, indicating that the anticancer activity was not affected by the EC coating. The main effect detected was that the ideal concentration of ETO could be decreased in the area of colon cancer. At this reduced concentration, the number of live normal cells could increase compared with the high level of dead normal cells that were detected at higher concentrations of free ETO, which makes our formula with a high concentration at the site of action safer.  
These results are logical and recommended. It has been reported that the ethylcellulose polymer is toxic to HeLa cells [42]. [PERSON] et al. [43] proved that ETO solid lipid nanoparticles have an IC\({}_{50}\) that was reduced to approximately 83% compared to that of  
Figure 8: Antiproliferative activity of free etoposide and ET–ETO microparticles at a drug–polymer ratio of 1:1 in MC-7 and Caco-2 cells. * _p_-value less than 0.05 (typically \(\leq\) 0.05) is statistically significant compared with cells treated with free drug.  
the free drug. Additionally, [PERSON] et al. [44] stated that lipid nanocapsules of ETO lowered the IC\({}_{50}\) of ETO from 100 to 2.5 uM in H209 cells, thus reducing the IC\({}_{50}\) of ETO to approximately 4-30-fold that in glioma cell lines [45]. This reduction in IC\({}_{50}\), which was also confirmed by [PERSON] et al. [46], effectively resolved the drug tolerance of MCF-7/ADR cells through a 4.3-fold reduction in IC\({}_{50}\) using an siRNA polyamidoamine complex. The suggested formulation (freeze-dried ET-ETO microparticles) provides very fast release at pH 7.4 (at the simulated duodenal pH) and low cytotoxic activity compared to free ETO against the tested cell lines used in their study. Based on these advantages, our findings are in agreement with those of [PERSON] et al. [47], who demonstrated that the use of targeted epichlorohydrin crosslinked Portulaca-alginate beads for colon cancer showed an enhancement in the antitumor activity as well as a reduction in the toxicity for some neoplastic drugs.