Development of polymeric micro/nanoformulates for the controlled and/or targeted release of antimicrobial peptides

With the dramatic rise in antibiotic resistance, including untreatable infections of multi-resistant bacteria and microbial biofilm-related diseases, there is no doubt that new effective antimicrobials or novel delivery systems for antibiotic compounds are urgently needed. Antimicrobial peptides (AMPs) represent a promising class of novel antimicrobials active against a wide range of microorganisms; despite an excellent antibiotic activity, their therapeutic potential is currently limited by their poor bioavailability, potential for systemic toxicity and sensitivity to protease degradation. The encapsulation of AMPs in polymeric carriers could protect them from degradation and secure their transport and delivery to the specific site of action at a controlled and tunable rate, rendering them effective and powerful antimicrobial tools. The present PhD project has been carried out in the interdisciplinary field of nanomedicine, with the aim of developing chitosan nanoparticles for the controlled delivery of the antimicrobial peptide Temporin-1b, which proved a strong antimicrobial activity against nosocomial multidrug-resistant Gram-positive bacteria. Chitosan (CS) was selected for nanoparticles (NPs) development due to its biocompatibility and biodegradability, fundamental characteristics in various biomedical applications. Due to the variability of commercially available batches of CS and to the strict dependence of its physical-chemical and biological properties to its structural features, commercial CS was fully characterized for its molecular weight (108 kDa, Mw/Mn 2.4) and deacetylation degree (~92%), key parameters for the development of CS NPs. Lysozyme (LZ) was encapsulated into chitosan nanoparticles as the first proteic model for cationic AMPs, as it is a relatively small protein (~14 kDa) with an optimal pH in the range of 6-9 and an isoelectric point near 9.2 (similarly to cationic AMPs), it exerts an antimicrobial activity against Gram-positive bacteria and it is considered to be the precursor of AMPs. LZ-loaded nanoparticles with good dimensional features (mean diameter 159 ± 24 nm) were successfully prepared by means of a mild ionic gelation technique. LZ loading in the NPs was up to 8% and the release kinetic studies showed that up to 20% of the loaded enzyme was slowly released over 3 weeks, in a controlled and sustained manner. The developed formulations showed a full in vitro cytocompatibility towards mammalian cells and LZ-loaded nanoparticles exhibited a good antimicrobial activity on Staphylococcus epidermidis, selected as a model Gram-positive pathogen. Renin substrate I (RSI) was then employed as an improved fluorogenic peptidic model for cationic peptides. In fact, despite the lack of an antimicrobial activity, RSI shares similar features with cationic AMPs (e.g. Temporin-1b), such as its small dimensions (8 amino acids), positive charge (+2) and hydrophobicity (75% of hydrophobic residues). The formulation parameters set for LZ were applied to the encapsulation of different amounts of RSI into CS nanoparticles and eventually tuned and optimized for maximum RSI encapsulation efficacy and loading. RSI encapsulation efficacy was raised to almost 100% by adjusting the pH of the CS solution from 3 to 5, thus promoting the hydrophobic interaction between CS backbone and peptide. RSI release kinetic from the NPs in vitro was evaluated: after a first equilibration time, all the formulations displayed a progressive linear release of the peptide. Through a mathematical modeling evaluation of RSI release profiles, Fickian diffusion was proposed as the peptide release mechanism from CS nanoparticles in vitro, dependent on the amount of loaded RSI and on the NPs radius. Finally, the optimized parameters set for the model peptide RSI were applied to the encapsulation of the AMP Temporin-1b (T-1b) into CS nanoparticles, thus reducing the waste of expensive AMPs during the initial research phases. T-1b-loaded CS nanoparticles with good dimensional features were prepared; T-1b EE% in the formulations was up to 75% and the release kinetic studies highlighted a linear release of the peptide from the NPs in the experimental conditions, confirming the results obtained from RSI-loaded CS NPs, selected as a model. Temporin-1b toxicity towards mammalian cells in vitro was compared to that of T-1b-loaded nanoparticles, proving the capability of the polymeric carrier to significantly reduce the peptide toxicity. Finally, the antibacterial activity of the developed nanoparticles was tested against S. epidermidis. T-1b-loaded NPs proved a long term antibacterial activity statistically superior to that of plain CS nanoparticles and plain T-1b, active in the initial incubation times. The antimicrobial NPs developed in this study seem to match the requirements of the “ideal” antibacterial delivery system: the bactericidal activity exerted by the nanocarrier itself (CS NPs) ensures an initial burst activity, markedly reducing the starting inoculum; afterwards, the linear release of T-1b from CS NPs secures a further reduction of viable bacterial counts, preventing the regrowth of the residual cells and ensuring a long-lasting antibacterial activity. The proposed AMPs-loaded CS nanoparticles could find their applications in different critical field of modern medicine, such as the treatment of multidrug-resistance systemic or topical infections and the antimicrobial pre-treatment or treatment of medical devices-related infections, thus having a huge impact on the progress of antimicrobial therapies