2.8.2. Antitumor Efficacy Evaluation

### Experimental Design

Efficacy and toxicity studies were performed on female Swiss albino mice (7–8 weeks of age, about 20–25 g). Animals were maintained under controlled temperature and humidity with a 12 h dark/light cycle, with free access to food and water.

Mammary tumors were induced in mice using the Ehrlich ascites tumor (EAT) model [26]. EAT cells, obtained from the National Cancer Institute, Cairo, Egypt, were properly diluted (~2 × 106) and subcutaneously injected into the left mammary fat pad of mice. The tumor volume was evaluated until it reached ~100 mm3 (12 days post-inoculation). Tumor-bearing animals were then randomly assigned into four groups (n = 6) as follows: positive control group (untreated), FS suspension in 10% PEG 400 saline solution, FS-NS, and LF-FS-NS administered by IP injection. Treated groups received 7 doses on an alternate-day dosing regimen of either FS suspension or NS formulations equivalent to 30 mg FS/kg. A negative control group was included for comparison. At the end of the study, mice were sacrificed, and tumors were excised and washed with ice-cold phosphate buffer. Tumor weight was determined, and then tumors were divided into portions for further assessment.

### Assessment of Tumor Growth

The percentage change in tumor volume compared to baseline volume was determined as an indicator of tumor growth. Tumor volume was assessed twice weekly. A vernier caliper was used to measure the tumor length (major axis) and width (minor axis), and then tumor volume was calculated according to the following equation [26]:

$$\text{Tumor volume} = \text{length} \times \text{ (width)}^2 \times 0.5 \tag{5}$$

Tumor Biomarkers

Excised tumors were homogenized in phosphate buffer saline (pH 7.4) to make a final 10% tissue homogenate that was used for the quantitative determination of tumor growth biomarkers. ELISA kits were used to assess cyclin-D1 level (Mouse cyclin-D1 ELISA Kit, EIAal™, Waltham, MA, USA) as an indicator of proliferation rate and the anti-apoptotic BCl-2 protein (catalog no. CSB-E08855m). Quantification was performed according to the manufacturer's protocol.

The relative expression of Bax and caspase-3 genes in tumor tissues, using quantitative real-time reverse transcriptase polymerase chain reactions (qRT-PCR), was performed. Forward and reverse primer sequences for PCR amplification are shown in Table 1. The RT-PCR reactions were run in triplicate with signal collection at the end of each cycle. An internal housekeeping gene (GAPDH) was applied to normalize relative transcript levels. The comparative threshold cycle (ΔΔCt) method was used to determine sample differences.

**Table 1.** qRT-PCR primers used to evaluate gene expression levels of Bax and caspase-3 in tumor tissues.


### Histopathological Examination

Excised tumor specimens were fixed in 10% formalin for 24 h. Sections (5 μm thickness) were cut, stained with hematoxylin and eosin (H&E) stain for 5 min, dehydrated in alcohol, and mounted in Canada balsam prior to microscopical examination.

### 2.8.3. In Vivo Toxicity Study

The effect of NS formulations vs. FS suspension on general animal health during the treatment course was evaluated. Change in body weight was recorded on a weekly basis. Following sacrifice, the liver, kidney, and spleen were excised and fixed in 10% formalin for histopathological examination. Furthermore, liver and kidney functions were evaluated by measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine in comparison to healthy animals.

### *2.9. Statistical Analysis*

All experiments were conducted in triplicate, and results were represented as mean ± SD. Statistical analyses were performed using an unpaired Student's *t*-test and one-way analysis of variance (ANOVA) followed by a post-hoc Tukey's test for multiple comparisons using GraphPad Prism (Version 7.04, San Diego, CA, USA). The level of significance was set at *p* ≤ 0.05.

### **3. Results and Discussion**
