*4.10. In Vivo Mouse Study for Screening of Antitumor Activity and Toxicity* 4.10.1. Mice Preparation and Ethical Approval

Thirty male Swiss albino mice (weight range equaled 25–30 g, 6–8 weeks of age) were purchased from VACSERA (Helwan, Egypt) and placed in groups of six in plastic cages. The experiment was done in the institutional animal house at the Faculty of Pharmacy, Suez Canal University, at a temperature range equal to 23 ± 5 ◦C, and the animals had free access to their normal diet and drinking water. The protocol of this study obtained approval from the institutional research ethics committee (#202004MA1).

## 4.10.2. Induction of Skin Lesions

A 2 <sup>×</sup> 2 cm<sup>2</sup> dorsal skin area was shaved on all animals using a hair clipper 48 h prior to the experiment. To induce skin lesions in mice, one dose of DMBA, which acts as an initiator for skin tumors (100 µg in 200 µL acetone) [63], was injected subcutaneously into each mice [28]. After one week, there was an increase in the number of epidermal lesions (the lesions > 1 mm in diameter for each mouse [63]. The skin lesions were assessed first by skin morphology (lesion width and lesion length) and, also, by histological methods (thickness of the epidermis).

4.10.3. Regimen of Applying Metformin-Loaded Ethosomes

Each group contained 6 animals, and the selected optimal formula was applied topically on the dorsal region of the skin (10 mg/cm<sup>2</sup> of the affected area) per week for a total of 4 weeks [27,48]. The experimental groups are shown in Table 4.


**Table 4.** Experimental groups for the in vivo mouse study.

The topical empty gel contained distilled water and carbopol 974 p only. On the other hand, the topical empty ethosome gel contained all the ethosome components (distilled water, ethanol, isopropyl alcohol, lecithin and cholesterol) and carbopol 974 p without metformin.

The measurements of the diameters of the skin cancer lesions were standardized for evaluating the efficacy of the selected optimal gel formula. Each mouse lesion more than 1 mm in diameter was measured weekly until the end of the study. At the end of the study protocol, the final lesion diameters were determined, and then, the mice were anaesthetized and slaughtered [64]. Blood samples were taken by cardiac puncture and settled for 30 min before centrifugation and separation of the serum samples.

#### 4.10.4. Histopathological Methodology and Examination

Extracted skin specimens were fixed in 10% neutral-buffered formalin, then embedded in paraffin wax and sectioned by a microtome (at 4 µm) and processed for hematoxylin and eosin (H&E) staining (the sections were deparaffinized, rehydrated in alcohol, stained in Harris hematoxylin, rinsed in 95% ethanol, counterstained with eosin solution, dehydrated through 95% alcohol and cleared in xylene, followed by mounting). Light microscopy was used to examine the skin sections by an experienced pathologist (the nuclei, nucleolus and nuclear membrane were stained blue, and the cytoplasm and connective tissue were stained pink) [30]. Histopathological investigation for skin tumor specimens was performed to assess the efficacy of the different drug/vehicle formulations [63]. Thicknesses of six regularly spaced skin parts were measured using ImageJ software (NIH, Bethesda, MD, USA). The average of the measured parts was calculated for every tissue specimen.

#### 4.10.5. Toxicological Screening

For testing any possibility of hepatic or renal toxicity due to the systemic absorption of the gel formula, a histopathological investigation was done for the liver and kidney specimens. The tissue samples were fixed in neutral-buffered formalin and processed for H&E staining and examination under light microscopy by an experienced blinded pathologist. In addition to the histopathological examination, the serum samples were directed for estimation of the liver enzymes (ALT and AST) and serum creatinine, urea and albumin.

#### *4.11. Statistical Analysis*

GraphPad prism was used to apply the statistical tests to the current data. Data were quantitative in nature and demonstrated in the form of the mean ± SD and analyzed using one-way ANOVA, as one factor (treatment regimen) was influencing the study groups. Bonferroni's test for multiple comparison analysis was at *p* < 0.05.

#### **5. Conclusions**

The topical application of ethosomal gel of metformin has a significant effect on treating chemically induced skin cancer in mice. This was shown by using the Box–Behnken design of "a three-level three-factor" to present a high percent of the EE%, minimum vesicle size, maximum ZP and high DR%.

Adding isopropyl alcohol with ethanol to form ethosomes increased the ability of ethanol to solubilize lecithin, which led to an increased stability and effectiveness of the ethosomes vesicles. Isopropyl alcohol also decreased the particle size of the vesicles, which increased the EE% and allowed the metformin to be released for an extended period. This is the goal of our research. Finally, a lower lecithin, high ethanol and isopropyl alcohol and moderate cholesterol obtained an enhancement of the permeation rate. Hence, the current findings may open up an avenue for future formulations for metformin as a therapeutic tool in fighting skin cancer.

**Author Contributions:** Conceptualization: O.M.S., T.M.H. and S.G.; Data Curation: I.A.M., T.M.H., S.G., S.A.Z., M.E.-S. and O.M.S.; Formal Analysis: I.A.M., S.A.Z. and O.M.S.; Software: I.A.M., T.M.H. and S.G.; Methodology: I.A.M. and O.M.S.; Resources: I.A.M. and O.M.S.; Validation: I.A.M. and O.M.S.; Visualization: T.M.H., S.G., S.A.Z., M.E.-S. and O.M.S.; Writing the Original Draft: I.A.M. and Writing, Review and Editing: T.M.H., S.G., S.A.Z., M.E.-S. and O.M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded partly by the AlMaarefa University researchers supporting program, AlMaarefa University, Riyadh, Saudi Arabia, grant number MA-006.

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of Faculty of Pharmacy, Suez Canal University (protocol code 202004MA1 on 12 April 2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors are thankful to Medical Union Pharmaceuticals (Abu Sultan, Egypt) for kindly providing the metformin hydrochloride powder, and also, we thank Future Pharmaceutical Company for providing the carbopol 974p powder.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

