Microparticulate and nanoparticulate systems as drug delivery vehicles for antimicrobial peptides targeting lung infections

Abstract

The increasing misuse and overuse of antibiotics have given rise to the health threat of antimicrobial resistance. To combat this urgent crisis, antimicrobial peptides (AMPs) have emerged as promising candidates due to their unique ability to disrupt bacterial cell membranes, reducing the development of resistance and exhibiting a broad spectrum of activity. This research study focused on the development of vehicles suitable for the delivery, especially through the lung, of three selected peptides, namely WLBU2, LL37 and hLF1-11. Despite the promise of AMPs, their susceptibility to in vivo proteolytic enzymes and high manufacturing costs have limited their clinical application. Localized drug administration, particularly via the pulmonary route, offers a targeted and efficient means of treating respiratory infections while minimizing systemic exposure. Dry powder inhalation emerged as the optimal vehicle for delivering AMPs directly to the lungs, enhancing their antimicrobial, anti-biofilm, and mucolytic efficacy. In this study it was demonstrated the potent antibactericidal activity against P. aeruginosa of these peptides with over 99% inhibition in just 2 minutes for WLBU2, while LL37 took 8 minutes. hLF1-11 exhibited moderate bactericidal effects in 2 minutes. The initial phase of this research project involved the manufacturing of spray-dried powders containing AMPs. A Design of Experiments approach was used to identify the influence of various process parameters on the spray-dried formulations. These consisted of mannitol and leucine for WLBU2, and trehalose and leucine for LL37 and hLF1-11. In vitro studies confirmed the safety and cytocompatibility of these formulations and peptides. Bactericidal activity assessments demonstrated the potency of WLBU2 and LL37, while hLF1-11 exhibited promising inhibition. Similar findings were also observed for the spray-dried formulations. Biofilm-associated infections, common in cystic fibrosis patients, were also targeted. Preliminary studies showed a reduction in bacterial biomass for WLBU2 and LL37, although further investigations are needed to determine the concentration needed to achieve the same effect once peptides are formulated. Circular dichroism studies revealed a strong interaction between AMPs and bacterial membrane-mimicking molecules, resulting in a conformational change of the peptides from unstructured to alpha helix. Moreover, for WLBU2 microparticles the intensity of this interaction was even more pronounced. Future research will explore these aspects for LL37 and hLF1-11 formulations. To address specific challenges associated with P. aeruginosa infections in cystic fibrosis patients, polymeric nanoparticle systems were explored, and two manufacturing processes were applied and compared. LL37, WLBU2, and hLF1-11 were encapsulated in nanoparticles of PLGA either by nanoprecipitation or nanoprecipitation assisted by microfluidics. The microfluidic technique led to particles in size of 200 nm, with AMPs encapsulation efficiency of about 30 % compared to the 10 % obtained with nanoprecipitation where particles were also > 250 nm. This was due to the inherent characteristics of this production method that allow the mixing processes to be controlled accurately and reproducibly. However, in vitro cell studies raised cytotoxicity concerns, requiring further investigation and optimization. This research has provided valuable insights into the potential of spray-dried and nanoparticle-based AMP formulations. Further studies, especially from a biological point of view regarding the antibacterial activity of the proposed systems, are essential to gain a comprehensive understanding of how effectively they can be utilized in the fight against bacterial infections and to make a meaningful contribution to the global battle against antimicrobial resistance. Additionally, addressing the specific challenges associated with cystic fibrosis-related infections remains a critical aspect of this research.