Schibeci, Martina (2024) Cutting-edge antimicrobial strategies: bacterial nanocellulose for the sustainable delivery of cryptic antimicrobial peptides identified in the human proteome. [Tesi di dottorato]

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Abstract

Over the past few centuries, the use of antibiotics allowed the treatment of infectious diseases caused by bacteria, fungi and viruses, significantly extending life expectancy. Unfortunately, their misuse has contributed to the alarming rise of AntiMicrobial Resistance (AMR), resulting in increased medical costs and mortality rates. In response to this challenge, Antimicrobial Peptides (AMPs) are emerging as promising alternative antimicrobial agents, since their administration typically does not promote the selection of resistant bacterial strains. In this Research project the sequence of the human precursor protein Matrix Metallopeptidase-19 (MMP-19) has been screened to identify cryptic AMPs. This led to the production of three novel AMPs, named r(P)YLL19, r(P)YLL33 and r(P)PRT33. These peptides demonstrated potent antimicrobial and anti-biofilm activities against skin, food-borne and cystic fibrosis pathogens, both independently and in combination with antibiotics, while exhibiting favourable biocompatibility and anti-inflammatory effects on eukaryotic cells. Additionally, the substitution of L-amino acids with D-amino acids in the case of r(P)YLL19 peptide led to the production of synthetic D(P)YLL19 peptide endowed with enhanced killing selectivity and biocompatibility. Since proteolytic degradation significantly limits the applicability of AMPs, non-peptidic carriers have been extensively employed in stable antimicrobial formulations, supporting the translation of AMPs to the market. Based on the growing emphasis on sustainable industrial processes, this project validates the employment of Bacterial Cellulose Nanoparticles (BCNPs) for the sustainable delivery of AMPs. BCNPs have been obtained through an optimized and reliable enzymatic hydrolysis characterized by low incubation times and by the possibility to recycle enzyme molecules. Furthermore, it was demonstrated that specific cellulose-producing strains affect the colloidal properties of BCNPs obtained by both enzymatic and acidic hydrolysis. BCNPs produced by enzymatic hydrolysis were successfully functionalized through the adsorption of r(P)ApoBLPro peptide, an AMP previously identified in the human precursor protein Apolipoprotein B100. The resulting system showed antimicrobial activity and good cytocompatibility properties. To further optimize BCNPs functionalization, MMP-19-derived peptides have been encapsulated in a Rapeseed oil-in-water Pickering emulsion by using unmodified BCNPs as the sole stabilizing agent. This approach resulted in a stable, active formulation requiring minimal AMPs doses and also providing a sustainable and versatile system to efficiently deliver natural AMPs without further chemical modifications.

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