In the last decade, the incidence of bacterial skin infections has been increasing at an alarming pace, establishing an important clinical and financial burden to health care systems [1]. The invasion of bacteria, such as Staphylococcus aureus, Cutibacterium acnes (formerly known as Propionibacterium acnes), Staphylococcus epidermidis and Streptococcus pyogenes, is the main cause of skin infections, which can be classified as localized superficial infections, like erysipelas, impetigo, acne folliculitis, rosacea, infected eczema, and deep tissue infections, such as cellulitis [2]. Among these infections, acne vulgaris is considered one of the most common skin diseases encountered worldwide, particularly affecting younger people [3]. Although it is not considered a typical infectious disease, it is characterized by an evident overgrowth of bacteria, like C. acnes, S. aureus and S. epidermidis [4], [5], [6]. C. acnes overgrowth triggers innate immune system activation, leading to cutaneous inflammation, follicular hyperkeratinization, lipogenesis, microcomedogenesis, and skin lesions of various morphologies, ranging from comedones, papules, and pustules to nodules and cysts [7].

An important aspect linked to skin bacterial infections concerns the alarming global spread of multidrug-resistant strains, which makes these pathologies difficult to treat, posing further a global threat to public health [8]. Particularly, the abuse or the misuse of antibiotics has contributed to the diffusion of increasing number of drug-resistant S. aureus and C. acnes strains, limiting treatment success [9].

Currently, topical administration of antimicrobial molecules through conventional dosage forms (such as creams, ointments, gels, or sprays) represents the first approach for the treatment of skin bacterial infections, owing largely to its advantage of minimizing drug systemic exposure. However, topical formulations present some drawbacks related to the difficulty to reach adequate drug concentration at the infected site [10]. This in turns requires multiple administrations, thus impairing patient compliance and worsening the antibiotic-resistance phenomenon [11]. The encapsulation of anti-infective agents into nanocarriers can represent an innovative approach useful to tackle the mentioned problems as well as to improve the therapeutic efficacy of anti-infective agents. In particular, peculiar characteristics of these drug delivery systems, like carrier size and surface charge, controlled release, the ability to interact with the main components of the skin, can allow to dramatically improve skin accumulation of the delivered drugs [12]. Additionally, multiple interactions of the nanosystems with the bacterial cell can promote cell wall and membrane disruption, membrane fusion and damage of bacterial intracellular components, thus improving the antimicrobial activity [13].

Azithromycin (AZT) is a macrolide antibiotic targeting the 50S bacterial ribosomal subunit, which inhibits protein synthesis, thus hindering the growth of bacteria [14]. Although AZT is mainly administered by oral and intravenous route, for the treatment of skin infections topical antibiotic therapy is crucial to avoid high dose and side effects [15]. Until now, only few papers have reported the use of nanosystems containing AZT for the topical treatment of skin infections. Particularly, Rukavina and colleagues [16] developed different types of AZT-loaded liposomes (conventional, deformable and containing PEG) to locally treat skin infections caused by methicillin-resistant S. aureus (MRSA) strains and demonstrated that all the prepared liposomes retained the drug inside the skin more efficiently than the control. Our recent study reported the preparation of AZT based microemulsions, which were able to guarantee a prolonged drug release and to promote drug accumulation inside the porcine skin [17]. In the current study, for the first time two different types of AZT-loaded vesicles, i.e., liposomes and niosomes (LP and NS) are proposed as useful nanocarriers for the treatment of skin bacterial infections and acne. Furthermore, to the best of our knowledge, this is the first study in which AZT-loaded LP and NS were prepared with different excipients by employing two different manufacturing methods (thin film hydration and ethanol injection) allowing to evaluate the influence of formulation and process factors on the physico-chemical and functional properties of the final products.

LP and NS are vesicles formed by self-assembly of phospholipids and non-ionic surfactants, respectively, which both present the ability to encapsulate hydrophilic and hydrophobic drugs [18], [19]. LP also possess the unique property of being biocompatible since their lipid bilayer structure mimics cell membranes and allows fusion with bacterial membranes [20]. However, one of the main limitation of LP regards their chemical instability linked to phospholipid hydrolysis or oxidation [21]. On the other hand, NS are generally characterized by higher chemical stability and longer shelf-life, intrinsic skin penetration enhancing properties and lower costs due to the availability of ingredients with reasonable cost compared to phospholipids [21].

Briefly, the main steps of this work were: (1) to prepare LP and NS with different excipients through the thin film hydration and ethanol injection methods; (2) to characterize them in terms of size, polydispersity index, ζ potential, encapsulation efficiency, morphology, nanomechanics and stability; (3) to investigate LP and NS ability to release the drug and to promote its accumulation within porcine skin; (4) to select the best formulations and to evaluate their antimicrobial activity and safety on fibroblast cells.