# Positively Charged Lipid as Potential Tool to Influence the Fate of Ethosomes
###### Abstract  
Ethosomes(r) are one of the main deformable vesicles proposed to overcome the stratum corneum. They are composed of lecithin, ethanol and water, resulting in round vesicles characterized by a narrow size distribution and a negative surface charge. Taking into account their efficiency to deliver drugs into deeper skin layers, the current study was designed to evaluate the influence of different lipids on the physico-chemical features of traditional ethosomes in the attempt to influence their fate. Three lipids (DOPE, DSPE and DOTAP) were used for the study, but only DOTAP conferred a net positive charge to ethosomes, maintaining a narrow mean size lower than 300 nm and a good polydispersity index. Stability and in vitro cytotoxic studies have been performed using Turbiscan Lab analysis and MTT dye exclusion assay, respectively. Data recorded demonstrated the good stability of modified ethosomes and a reasonable absence of cell mortality when applied to human keratinocytes, NCTC 2544, which are used as a cell model. Finally, the best formulations were selected to evaluate their ability to encapsulate drugs, through the use of model compounds. Cationic ethosomes encapsulated oil red o and rhodamine b in amounts comparable to those recorded from conventional ethosomes (over 50%). Results recorded from this study are encouraging as cationic ethosomes may open new opportunities for skin delivery. ethosomes; cationic ethosomes; drug delivery systems; surface charge; cutaneous administration +
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vesicles that were able to more easily permeate through skin layers [14]. Ethosomes(r), first introduced by [PERSON] et al. [15] are one of the main known deformable systems for topical use. Ethanol, present in small quantities in their structure, acts as permeation enhancer, reversibly modifying the skin structure [16] to facilitate the passage of active compounds through skin. In detail, [PERSON] et al. demonstrated that using greater amounts of ethanol during the preparation of the sample, the realization of micellar systems can occur, but in the case of amounts lower than 50% \(w/v\), the vesicular bilayered structure was retained [15]. Although deformable vesicles have been shown to be able to increase the transcutaneous penetration of drugs both in vitro and in vivo [17; 18; 19; 20], the influence of surface charge modifications on permeation still represents an open question [20; 21; 22]. Indeed, even if the effectiveness of negatively charged liposomal systems has already been reported in many studies [19; 23; 24], other authors have assumed and demonstrated that the skin permeation of molecules loaded in positively charged vesicles, obtained using, for example, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), was greater than that recorded in the case of negatively charged ones [25; 26]. In particular, the electrostatic interaction between positively charged vesicles with skin lipids having negative charge seemed to enhance penetration through skin. An interesting study proposed by [PERSON] and co-workers [27] proved that cationic liposomes were able to reach an average relative penetration depth of around 70% of hair follicle length. In this work, the authors obtained cationic nanosystems made up of dimyristoylphosphatidylcholine, N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate, and cholesterol (50:10:40 mol%), able to interact with negative charge of the hair surface by ion-exchange, reaching an high local concentration. Considering that hair follicles can play the role of reservoir for topically applied molecules, these skin appendages could represent a target of particular interest for transcutaneous delivery of active molecules. For this reason, the great ability of cationic ethosomes to permeate skin through hair follicles needs to be taken into account and further investigated. The aforementioned hypotheses have prompted us to realize a new system having similar composition with respect to classical ethosomes, already known as good skin permeating systems.