# Positively Charged Lipid as Potential Tool to Influence the Fate of Ethosomes
## 3 Results and Discussion
### Characterization of Cationic Vesicles Made of Different Amount of DOTAP  
Starting from the interesting results obtained for formulation D, further investigations were performed to test the influence of variable amounts of DOTAP in the main features of the sample and to find the best concentration to be used for subsequent in vitro and in vivo studies. For this reason, the concentration of cationic lipid, expressed as molar ratio (PL90 G:DOTAP) was both progressively increased (formulations E-H) and reduced (formulations I-M) compared to the amount reported for formulation D (PL90G:DOTAP, 10:1) and further characterization studies have been performed using Zetasizer Nano ZS. As can be seen in Table 3, the increase in DOTAP amount led to an increase in vesicle sizes. Polydispersity index values also increased compared to that recorded from formulation D and this was probably due to the simultaneous formation of micellar systems following the accumulation of lipids that were not structured in the carrier [42]. The mean sizes of vesicles also increased, overcoming 500 nm in size, which was too high a value to consider these carriers for topical cutaneous administration [37]. Figure 1: Delta transmission (\(\Delta\)T) and delta backscattering (\(\Delta\)BS) profiles of cationic ethosomes made of Phospholipon 90G and DOTAP (10:1 molar ratio). The figure is representative of three different experiments performed for the sample. Data are reported as a function of time (0–3 h) and sample height (mm). On the other hand, the reduction in the amount of DOTAP used during the preparation of formulations I-M showed good values of size and polydispersity index, but a gradual reduction in the net positive surface charge of the vesicles congruent with a reduction in the amount of positive lipid added during the preparation of samples. The increase in the zeta potential values towards more positive values as a function of the increasing amount of DOTAP added confirmed that the cationic lipid molecules were disposed on the surface of the nanovesicles. Despite the reduction of zeta potential values related to the reduction in the addition of cationic lipids, all investigated samples showed values beyond the threshold of +30 mV, commonly considered suitable for guaranteeing repulsion between vesicles thus avoiding aggregation phenomena [40]. In fact, the values needed to avoid destabilizing phenomena are questioned due to the great influence of several other parameters to be considered, such as the solvent used, the concentration of ions, and functional groups of the surface [40]. For this reason, further stability studies have been performed. The stability kinetics profiles of all cationic endosomes (formulations D-M) were investigated using Turbiscan Lab analysis and the results were compared. As can be seen in Figure 2, the addition of different amounts of DOTAP did not result in a marked variation of TSI values compared to those recorded for formulation D, which also reported \(\Delta\)T and \(\Delta\)BS profiles within an excellent stability range (Figure 2). These results suggested that no relevant instability phenomena were to be expected for the analyzed samples.