*4.8. Morphological Examination of the Optimal Metformin-Loaded Ethosomes Formula*

The morphology of the optimal metformin-loaded ethosome formula (#13) in the gel preparation ensured that the vesicles were still formed. The gel was characterized by using TEM (TEM-1010, Tokyo, Japan) [61]. After sample preparation, it was dropped onto the surface of a copper grid coated with carbon. Each sample was left to dry in order to permit ethosomes to adhere to the carbon substrates. For staining, we applied a drop of 1% aqueous phosphotungestic acid dye to the grid, which was then air-dried for 2 min after removing excess dye with a piece of filter paper. The TEM was then used to examine and visualize the stained sample.

## *4.9. Thermal Analysis of Optimal Metformin-Loaded Ethosomes Formula*

The thermal analysis of the optimal metformin-loaded ethosomes formula was studied by utilizing differential Scanning Calorimetry (Shimadzu, DSC 60, Kyoto, Japan). Five milligrams of each sample were added to an aluminum pan. Each sample was heated from room temperature to 300 ◦C at a heating rate equal to 10 ◦C/min under nitrogen flowing at a rate of 20 mL/min to prevent oxidation of the sample [62]. Pure metformin, optimal metformin-loaded ethosome formula and empty formula (excipient) thermograms have been compared.
