Dottorato in SCIENZE FARMACEUTICHE

Titolo della tesi: Liposomes as a versatile tool for the delivery of natural antimicrobials

Antibiotics are indispensable pharmaceuticals to treat bacterial infections. However, they have been overused in clinic, agriculture and animal production setting, generating a strong selection pressure over bacterial species. This has led to the emergence and dissemination of antimicrobial resistant strains among humans, animals and the environment, culminating in the rise of the global health problem of antibiotic resistance, which requires immediate and urgent actions to be fought. Consequently, significant funding has been allocated for the development of new and effective strategies to expand the panel of therapeutic tools already available. Nevertheless, the development of new antibiotic drugs is a laborious process which cannot keep up with the rising rates of drug resistant bacteria. In this perspective, an appealing alternative to the discovery of new therapies could be the use of bioactive compounds derived from biomass waste, thus representing an example of circular economy in which biomass waste are converted in valuable products, minimizing waste production. Among all bioactive compounds recoverable, polyphenols exhibit beneficial effects on human well-being, such as antimicrobial activity against both Gram-positive and Gram-negative bacteria. Nevertheless, in some cases the polyphenols potential therapeutic use is limited by their adverse pharmacokinetic features resulting in a very low absorption rate after systemic administration in human body. In order to overcome these limitations, many engineered nanoparticles can be applied as drug delivery systems due to the possibility of engineering them for multiple purposes. Among all the nanoparticles developed in the last decades, liposomes are the most widely used drug delivery systems thanks to their versatility, low toxicity, biocompatibility and biodegradability. In this scenario, this work of thesis aimed to develop liposomes as delivery systems of natural antimicrobial polyphenols, both as single biocompound and as phytocomplexes, thus improving their pharmacokinetic profiles and enhancing their activity against target bacteria examined. Polyphenols investigated in this thesis were obtained from agri-food by-products and waste, such as olive leaves and orange peels. Liposomes were formulated with natural phosphocholines (DMPC, DPPC, DOPC) and cholesterol, in presence or absence of synthetic cationic amphiphiles, which should enhance the interaction between liposomes developed and target bacteria strains. In particular, two different cationic amphiphiles were used: 1) a galactosylated amphiphile (GLT1) characterized by the presence of a galactose residue, which should enhance the interactions toward lectins and sugar-protein transporters expressed on bacterial membrane; 2) the allyl-hexadecyl-dimethyl-ammonium iodide, characterized by a polar head composed by a quaternary nitrogen and a hydrophobic alkyl chain, which should be able to enhance the electrostatic interaction between cationic liposomes and negatively charged bacteria. The overall composition of the formulations was modulated in order to obtain vesicles with dimensions around 100 nm, a good polydispersity index related to the homogeneity of the systems, variable ζ-potential values, good stability with respect to aggregation phenomena and optimal entrapment efficiencies of polyphenols inside the liposomes. The antimicrobial activity of polyphenols, free and entrapped in liposomes, was examined in vitro against different bacteria pathogens, both Gram-positive and Gram-negative strains, such as Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella oxytoca.