## 2 Materials and Methods
### Formulation and Evaluation of the Topical Creams That Contain ZnNPs
#### 2.3.2 Evaluation of the Creams  
**Microscopic analysis of emulsions**  
The microscopic images of cream formulations were taken after 24 h of storage using a KRUSS optical microscope equipped with a photo-digital camera (Nikon, Coolpix P 5100 (Tokyo, Japan)). **Organoleptic characteristics**  
The organoleptic characteristics of the creams (homogeneity, texture, color, stability, odor) were observed. **Determination of pH**  
One gram of each cream formulation was dispersed in 100 mL of deionized water and stored for 2 h. pH of the formulations was determined at room temperature using a \begin{table}
\begin{tabular}{l l l l l l l} \hline \hline \multirow{2}{*}{**Ingredients**} & \multicolumn{6}{c}{**Samples**} \\ \cline{2-7}  & **S1** & **S2** & **S3** & **S4** & **S5** & **S6** \\ \hline Beeswax (g) & 2 & 2 & 2 & 2 & 2 & 2 \\
10\% (\(\
u/\
u\)) Span 80 solution in olive oil (mL) & 2.4 & 2.4 & 2.4 & 2.4 & 2.4 & 2.4 \\ Glycerol (mL) & 2.7 & 2.7 & 2.7 & 2.7 & 2.7 & 2.7 \\
20\% Tween 80 solution (mL) & 3 & 3 & 3 & 3 & 3 & 3 \\ Gelatine (g) & 1 & 1 & 1 & 1 & 1 & 1 \\
3\% chitosan solution (g) & 6 & 6 & 6 & 6 & 6 & 6 \\ VLSENPs (g) & - & 0.2 & - & - & - & - \\ ZnNPs (g) & - & - & 0.2 & 0.3 & 0.4 & 0.5 \\ Distilled water (mL) & 22.2 & 22.0 & 22.0 & 21.9 & 21.8 & 21.7 \\ \hline \hline \end{tabular} The amounts from each ingredient required to obtain these creams have been established after preliminary tests.  
\end{table} 
Table 1: Composition of the topical cream formulations. HI8010 (Hanna instruments, Leighton Buzzard, UK) pH meter. For each cream, the pH was measured in triplicate. The mean values and the \(\pm\) standard deviations were calculated. **Determination of viscosity**  
The viscosity of the formulated creams was determined at room temperature with a PCE-RVI 1 ViscometerDeutschland GmbH viscometer using the spindle number 4. The stirring speed was set to 6 rpm. All viscosity measurements were performed in triplicate. The mean values and the \(\pm\) standard deviations were calculated. **Spreadability**  
The spreadability of the cream formulations was determined by the parallel plate method [33]. Two glass slides of 20/20 cm were selected. 1 g of sample was applied between the two glass plates. Upon the upper glass plate was placed a weight with the mass (m) of 100 g so that the cream between the two slides was pressed uniformly to form a thin layer. After one minute from the placing of the weight, the diameter of the sample (d) was measured. The spreading diameter of each cream formulation (cm) was measured in triplicate to obtain the average spreading diameter \(\pm\) standard deviation (SD). **Irritancy test**  
The testing of the skin irritation was carried out on five volunteers. A half gram of cream was applied on the forearm skin area of each volunteer for 24 h. After removal of the cream, the subjects were examined for 24 h, at 2 h intervals, to identify the occurrence of any erythema, redness, or sensation of itching. **'In vitro' evaluation of antibacterial activity**  
Antimicrobial activity testing was performed under the specific conditions for in vitro biological testing by exposing some samples to different microbial cultures. The test methods are selected and adapted according to the nature of the samples. The principle of these methods is based on the ability of molecules with antimicrobial properties to diffuse into the culture media used for microbial development and to interact with the microbial cells against which they exert an inhibitory effect or a microbicidal (bactericidal/fungicidal) activity. The antimicrobial activity of the cream samples (S1, S2, S3, S4, S5, S6) was tested against the bacterial species _Staphylococcus aureus_ (ATCC 25923), _Methicillin-resistant Staphylococcus aureus_ (MRSA ATCC 33591), _Escherichia coli_ (ATCC 25922) and the yeast species _Candida albicans_ (ATCC 90028), using the standardized Kirby-Bauer diffusion susceptibility method adapted for this type of sample.