Liposomes loaded with the pneumococcal endolysin MSlys: From in vitro characterization to ex vivo permeation across the tympanic membrane

Introduction: The increasing antibiotic resistance triggered interest in novel antimicrobials as well as delivery systems that allow a topical and targeted delivery. However, biological barriers, such as the skin or the tympanic membrane (TM), may hinder the success of the therapy. Drug permeation has been extensively studied in the context of transdermal delivery, but only recently started to be explored for transtympanic applications. Ex vivo Franz diffusion cell permeation tests have been used and validated for the permeation of compounds through the skin prior to in vivo studies, but their exploitation for transtympanic delivery is limited. Endolysins, peptidoglycan hydrolases derived from bacterial viruses, are attractive antimicrobials, with promising action against the otitis media pathogen Streptococcus pneumoniae. Liposomal systems, such as transfersomes or PEGylated liposomes, have been shown to enhance drug permeation across the TM. Here, we describe the in vitro characterization of the endolysin-loaded liposomal carriers as well as their ex vivo permeation through TMs. Objectives: The main objective was to develop a delivery system containing an endolysin for a targeted transtympanic treatment of otitis media. To achieve this, it was necessary: i) to encapsulate the endolysin into liposomes for a controlled delivery; and ii) to evaluate the transtympanic permeation ability of the formulations. Materials and Methods: Liposomes composed of 4 mM of L-alpha-lecithin and sodium cholate (5:1) (L:SC) or L-alpha-lecithin and PEG2000 PE (10:1) (L:PEG) loaded with the MSlys endolysin were prepared. The size, polydispersity index (PDI), zeta potential, stability, deformability, encapsulation efficiency, and in vitro MSlys release were determined. The cytotoxicity against fibroblasts and keratinocytes and the efficacy against pneumococcal planktonic and biofilm cells were also evaluated in vitro. Permeation studies were performed in Franz diffusion cells using porcine skin, sheep TMs, and cadaveric human TMs. ...