# Positively Charged Lipid as Potential Tool to Influence the Fate of Ethosomes
## 3 Results and Discussion
### Evaluation of Encapsulation Efficiency  
The ability of formulations D and I to encapsulate active compounds was also investigated by adding oil red O, rhodamine B or bromophenol blue salt as lipophilic, hydrophilic, and amphiphilic model drugs, respectively. First of all, further characterization studies have been performed to investigate the mean sizes, zeta potential and the stability of samples to assess whether they suffered significant changes and if they retained their suitability for topical use. It was interesting to note that in any case the addition of a probe, regardless of their chemical properties, caused a significant (\(p<0.001\)) reduction in vesicle sizes (Table 4), probably related to a rearrangement of the structure of the carrier. A slight increase in the polydispersity index values was recorded for the addition of oil red o, despite all values remaining below an acceptable threshold [47]. As in the case of the charge of cationic ethosomes (formulations D and I), a reduction of values was recorded proportionally to the reduction in size compared to empty formulations. Nevertheless, carriers loaded with one of the three probes still retained a net positive surface charge that was proportional to the amount of cationic lipid added during preparation. The stability of each sample was checked using Turbiscan Lab analysis (Figure S2). Formulations showed good stability profiles reporting \(\Delta\)Backscattering values in a range of \(\pm 1\) for any probe added in conventional ethosomes (Figure S2A) or into cationic ethosomal systems (Figure S2B,C) thus confirming that no creaming, flocculation or sedimentation phenomena occurred by the addition of model compounds into the realized systems.  
The new realized cationic systems also showed good abilities to encapsulate hydrophilic and lipophilic probes (above 50% of the amount added during the phase of preparation), comparable with those recorded from well-known drug delivery systems, i.e., conventional ethosomes [15; 34; 36]. In detail, formulations D and I seemed to encapsulate oil red o better than conventional ethosomes, probably due to a greater interaction between the hydrophobic model compound and the cationic lipid, i.e., DOTAP. Concerning the addition of rhodamine B, conventional ethosomes (formulation A) and cationic ethosomes made of PL90G:DOTAP 10:0.5 molar ratio (formulation I), reported very similar values, while a slight decrease was recorded for an increased amount of DOTAP (formulation D, 10:1 molar ratio). This effect was probably due to a greater bulky effect on the part  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Dye**} & \multirow{2}{*}{**Formulation**} & \multirow{2}{*}{**Mean Size**} & \multirow{2}{*}{**Polydispersity**} & **Zeta** & **Encapsulation** \\  & & **(nm)** & & **Index** & **Potential** \\  & & & & **(mV)** & **(\%)** \\ \hline \multirow{4}{*}{Oil red O} & A & 100 \(\pm\) 1 & 0.19 \(\pm\) 0.02 & \(-\)25 \(\pm\) 1 & 43 \(\pm\) 3 \\  & D & 112 \(\pm\) 3 & 0.29 \(\pm\) 0.01 & +38 \(\pm\) 2 & 55 \(\pm\) 2 \\  & I & 105 \(\pm\) 1 & 0.33 \(\pm\) 0.01 & +29 \(\pm\) 3 & 50 \(\pm\) 2 \\  & A of the lipid to the detriment of the aqueous core where the hydrophilic probe is likely to accumulate. Finally, regarding the addition of amphiphilic molecules, such as bromophenol blue, a drastic reduction of the amount encapsulated was recorded for both cationic and conventional ethosomes and once again this amount was lower for increasing amounts of DOTAP in the sample.