# Positively Charged Lipid as Potential Tool to Influence the Fate of Ethosomes
## 3 Results and Discussion  
### Physico-Chemical Characterization Studies  
A deeper investigation of the physico-chemical and technological formulation properties of a new drug delivery system represents both a fundamental and limiting step for the realization of an eligible formulation. The suitability of a nanocarrier for a specific administration route depends on different parameters. Indeed, mean sizes, size distribution, any surface charge and the stability of a sample over time are important factors that influence the pharmacokinetics of the realized system [26; 29; 30]. Especially in the case of cutaneous administration, these parameters are essential to define the permeation of the carrier or any delivered substance [20; 31; 32]. In this study, a new carrier for topical cutaneous administration was realized and characterized, taking into account the conventional composition of ethosomes, a well-known drug delivery system having advantageous properties for cutaneous administration [15]. The realization of the new system was made in the attempt to maintain the ability of conventional ethosomes to be used as skin drug delivery systems, modifying their surface charge to influence their fate. Conventional ethosomes owe their ability to permeate through the skin barrier to the elasticity conferred by the presence of ethanol in their structure [33; 34]. Moreover, data reported in the literature often showed a correlation between ethosomal size and the amount of lipids and ethanol in their structure. In particular, sizes of ethosomes decrease as the concentration of ethanol increases and soybean phosphatidylcholine decreases [15; 35]. Since size represents a very important feature for topical drug delivery systems, we chose conventional ethosomes with minimum amount of phospholipid 90G (1% \(w/v\)) and a high amount of ethanol (40% \(w/v\)) to propose this system as the control for our further studies. The threshold of 40% \(w/v\) ethanol was not exceeded because, as previously reported, these systems showed a certain instability in the presence of an amount of 45% \(w/v\) ethanol [36]. Analysis of dynamic light scattering was performed using Zetasizer Nano ZS and results, reported in Table 2, showed a narrow size distribution with dimensions lower than 300 nm for the conventional ethosomal (formulation A), thus indicating that they were small enough for topical administration and transcutaneous passage [37]. Ethosomes having conventional composition, also reported a net negative surface charge, which is essential to avoid aggregation phenomena and to ensure a good stability of sample over time [38]. The stability of conventional ethosomes was further investigated using Turbiscan Lab(r). Results of \(\Delta\)Transmission and \(\Delta\)Backscattering showed good stability profiles, within a range of \(\Delta\)Backscattering \(\pm\)1 (see Supplementary Materials, Figure S1), thus confirming the results obtained in one of our previous studies [36]. In fact, the ability of conventional ethosomes to interact with skin lipids and to improve the permeation of any delivered drug has been widely studied [16; 36; 39]. However, due to recent and conflicting discoveries concerning the influence of the surface charge on the transdermal flux and direction of delivered drugs [22; 26], new vesicular systems have been realized using three different lipids having positive charges in their structures, with the aim to investigate how much they influence the behavior of ethosomes. In this attempt, the first step regarded the design and characterization of a cationic ethosomal formulation. The composition of formulation A was retained and the amount of PL(r)O G was replaced with the same amount of a lipidic mixture between PL(r)O G and lipids with positive charge (10:1, molar ratio).