## 3 Results and Discussions
### Antimicrobial Activity against Gram (+) and Gram (\(-\)) Strains
\begin{table}
\begin{tabular}{c c c c c} \hline
**Samples** & **Bacteria** & **S. aureus** & **E.** _fecalis_ & **E. coli_ & **P. aeruginosa** \\ \cline{2-5}  & & & **Inhibition Zone \(\pm\) SD (mm)** & \\ \hline AA15\% & 20 \(\pm\) 0.12 & 20 \(\pm\) 0.11 & 13 \(\pm\) 0.11 & 9 \(\pm\) 0.30 \\ AA20\% & 23 \(\pm\) 0.15 & 21 \(\pm\) 0.15 & 15 \(\pm\) 0.15 & 10 \(\pm\) 0.12 \\ AA25\% & 22 \(\pm\) 0.20 & 20 \(\pm\) 0.11 & 20 \(\pm\) 0.12 & 11 \(\pm\) 0.30 \\ AALipo15\% & 22 \(\pm\) 0.14 & 22 \(\pm\) 0.10 & 18 \(\pm\) 0.20 & 9 \(\pm\) 0.11 \\ AALipo20\% & 26 \(\pm\) 0.20 & 24 \(\pm\) 0.13 & 19 \(\pm\) 0.14 & 10 \(\pm\) 0.20 \\ AALipo25\% & 20 \(\pm\) 0.30 & 20 \(\pm\) 0.20 & 16 \(\pm\) 0.12 & 11 \(\pm\) 0.12 \\ Van 30 \(\mu\)g & 18 \(\pm\) 0.40 & - & - & - \\ Amp 2 \(\mu\)g & - & 0 & - & - \\ Ctx 30 \(\mu\)g & - & - & 33 \(\pm\) 0.40 & - \\ Ctx 20 \(\mu\)g & - & - & - & 24 \(\pm\) 0.30 \\ \hline \end{tabular} Legend: AA15\%—Azeelaic acid solution 15%, AA20%—Azeelaic acid solution 20%, AA25%—Azeelaic acid solution 25%, AALipo15%—I liposomes loaded with AA in a concentration of 15%, AALipo20%—Iposomes loaded with AA in a concentration of 25%, Van—Vancomycin, Amp—Ampicillin, Ctx—Cefftraxone, Ctz—Ceffazidime.  
\end{table} 
Table 2: Antimicrobial activity of AA and AALipo formulations against gram (\(+\)) (_Staphylococcus aureus_ and _Enterococcus faecalis_) and gram (\(-\)) strains (_Escherichia coli_ and _Pseudomonas aeruginosa_) expressed as diameter of inhibition zone. is maintained in the pilosebaceous follicle. A change of the microbiome composition may lead to a disturbed skin barrier and inflammation. Studying cutaneous samples, [PERSON] et al. observed that when _P. acnes_ was present, _Pseudomonas_ species typically were not, and vice versa [46]. Interestingly, antibiotic treatment for acne that decreases _P. acnes_ colonization on the skin may also result in Gram-negative folliculitis caused by _Pseudomonas_[46]. [PERSON] et al. recently proposed that _P. acnes_ strains may be implicated in antimicrobial defense pathways by triggering a local increase in the autophagic activity of keratinocytes [47]. However, it is generally accepted that advanced formulations of AA (hydrogel, micro- or nano-emulsions, etc.) possess enhanced pharmaceutical bio-availability compared to conventional cream based on AA [48]. Cell Viability and Cytotoxicity (MTS Assay) of Normal Human Dermal Fibroblast Culture under Different Treatments with Liposomal Formulations  
The percentage of cell viability based on trypan blue exclusion upon different treatment with liposomal formulations is presented in Table 3.  
The MTS assay was employed to evaluate the potential cytotoxicity of both empty liposomes (Lipo) and different liposomal formulations with AA, in comparison to the untreated \"wound\" (CTRL sample) using NHDF cells (Figure 5). Our results showed that in the case of AA, there is a concentration-dependent decrease in cell viability. At the highest concentration tested (25%), a significant decrease of cell  
Figure 5: Cytotoxicity assay of different concentration of azelaic acid and liposomes loaded with azelaic acid (AALipo15%, AALipo20%, AALipo25%) compared to the untreated sample (CTRL) and the positive control (Lipo) with respect to human dermal fibroblasts. All values were expressed as mean \(\pm\) SD. Samples (Lipo, AA15%, AA20%, and AA25%) were compared with CTRL (\({}^{\#}\)_p_\(<\) 0.01, \({}^{\#}\)_p_\(<\) 0.05), and liposomes loaded with different doses of azelaic acid were compared with Lipo sample (\({}^{\#}\)_p_\(<\) 0.01, \({}^{\#}\)_p_\(<\) 0.05). \begin{table}
\begin{tabular}{c c} \hline \hline
**Samples** & **\% Cell Viability** \\ \hline CTRL & 93.25 \(\pm\) 5.21 \\ Lipo & 94.15 \(\pm\) 3.50 \\ AALipo15\% & 93.35 \(\pm\) 4.90 \\ AALipo20\% & 94.11 \(\pm\) 8.11 \\ AALipo25\% & 94.48 \(\pm\) 5.24 \\ \hline \hline \end{tabular}  
Lipo—blank liposomes, AALipo15%, 20%, 25%—liposomes loaded with AA in concentration of 15%, 20% and 25% respectively; CTRL—no treatment.  
\end{table}